Quality control method of compound radix biondiellae stomachache capsule

Abstract

The application discloses a quality control method of compound radix bupleuri stomachache capsules, which comprises the following steps: (1) preparing a control sample solution by taking paeonolactone glycoside as a control sample; (2) preparing a test sample solution of the compound radix bupleuri stomachache capsules; (3) precisely sucking the control sample solution and the test sample solution respectively to carry out high performance liquid chromatography detection, wherein the high performance liquid chromatography conditions comprise the following steps: taking octadecylsilane bonded silica gel as a filler, taking acetonitrile as a mobile phase A, and taking 0.18-0.22% triethylamine phosphate solution as a mobile phase B, and then detecting by gradient elution. The application explores a method for quantitatively detecting paeonolactone glycoside by using high performance liquid chromatography, improves the standard level of the compound radix bupleuri stomachache capsules, and is more favorable for the quality control of the compound radix bupleuri stomachache capsules.

Description

Technical Field

[0001] This invention belongs to the field of drug quality control, and specifically relates to a quality control method for Compound Tianqi Stomach Pain Capsules. Background Technology

[0002] Chronic gastritis, gastric ulcers, and duodenal ulcers are common digestive system diseases, more prevalent in men than women. They are often triggered by factors such as emotional fluctuations, overwork, dietary imbalances, smoking, excessive alcohol consumption, and adverse effects of certain medications. The disease has a long course, with frequent relapses, and prolonged illness can lead to more complex pathogenesis.

[0003] Compound Tianqi Stomach Pain Capsules are made from Panax notoginseng, Corydalis yanhusuo (vinegar-processed), Cyperus rotundus (vinegar-processed), Evodia rutaecarpa (vinegar-processed), Ostrea gigas (calcined), Alum, Licorice, White peony root, Bletilla striata, Melia toosendan, Magnesium oxide, Sodium bicarbonate, and Belladonna extract. Its functions are to regulate qi and remove blood stasis, warm the middle jiao and strengthen the spleen, and astringe and stop bleeding. It is used for hyperacidity, stomach pain, gastric ulcers, duodenal bulb ulcers, and chronic gastritis.

[0004] Compound Tianqi Stomach Pain Capsules are listed in the Ministry of Health's Drug Standards, Volume 16 (Protected Varieties, Volume 1), WS3-B-3078-98. The standard's testing content is relatively limited, only including microscopic and test tube reactions, which is not conducive to product quality control. Furthermore, the current standard for Compound Tianqi Stomach Pain Capsules lacks a content determination item, hindering the improvement of its standards and accurate quality control.

[0005] To improve the quality standard control level of Compound Tianqi Stomach Pain Capsules, we conducted a systematic standard study and established a high-performance liquid chromatography detection method for paeoniflorin in Compound Tianqi Stomach Pain Capsules.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a quality control method for Compound Tianqi Stomach Pain Capsules. It explores a method for quantitative detection of paeoniflorin in Compound Tianqi Stomach Pain Capsules using high performance liquid chromatography, which improves the standard level of Compound Tianqi Stomach Pain Capsules and is more conducive to the quality control of Compound Tianqi Stomach Pain Capsules.

[0008] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0009] The purpose of this invention is to provide a quality control method for Compound Panax notoginseng Stomach Pain Capsules, comprising the following steps:

[0010] (1) Prepare a reference solution by taking paeoniflorin reference standard;

[0011] (2) Preparation of the test solution of Compound Tianqi Stomach Pain Capsules;

[0012] (3) Accurately pipette the reference solution and the test solution separately for high performance liquid chromatography (HPLC). The HPLC conditions include: using octadecylsilane-bonded silica gel as the packing material; using acetonitrile as mobile phase A and 0.2% triethylamine phosphate solution as mobile phase B, and performing detection by gradient elution.

[0013] Analysis of the formulation of Compound Tianqi Stomach Pain Capsules revealed that white peony root is used as an assistant herb in a relatively large dosage, possessing antispasmodic and analgesic effects (see Part I, White Peony Root, 2020 Edition of the Chinese Pharmacopoeia). The pharmacopoeia's content determination standard for white peony root stipulates that the paeoniflorin content should not be less than 1.6%. Based on the approximately 30% water extraction transfer rate of white peony root in other traditional Chinese medicine preparations, the paeoniflorin content in the finished product is estimated to be approximately 0.08%. Therefore, using paeoniflorin as an indicator to improve quality standards is feasible. However, apart from white peony root, the contents of other medicinal materials, calculated based on the input amount and the lower limit of their content, are all less than 0.05%, making quality control less meaningful.

[0014] Since the current standard for Compound Tianqi Stomach Pain Capsules does not include a content determination item, this invention studies and establishes a method for detecting the content of paeoniflorin in Compound Tianqi Stomach Pain Capsules using high performance liquid chromatography (HPLC). This method serves as a control standard for evaluating the quality of Compound Tianqi Stomach Pain Capsules, which is beneficial for improving the quality control of Compound Tianqi Stomach Pain Capsules.

[0015] A further approach involves gradient elution conditions including:

[0016]

[0017] A further proposed approach is to use a high-performance liquid chromatography (HPLC) detection wavelength of 230 nm, and the theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 9000.

[0018] A further proposed method involves a column temperature of 25–35°C and a flow rate of 0.9–1.1 ml / min.

[0019] In a further step, in step (2), the contents of Compound Tianqi Stomach Pain Capsules are taken, and hydrochloric acid solution is added to adjust the pH value to neutral. The contents are weighed, ultrasonically treated, cooled, and weighed again. The weight loss is replenished with hydrochloric acid solution, mixed, and filtered. The filtrate is measured, and water-saturated n-butanol is used for extraction. The filtrate is evaporated to dryness, and the residue is dissolved in methanol to obtain the test solution.

[0020] Compound Tianqi Stomach Pain Capsules contain a large amount of alkaline components, therefore the aqueous solution of the contents is alkaline. However, under alkaline conditions, paeoniflorin has poor thermal stability. In this invention, hydrochloric acid solution is used as the extraction solvent to create a neutral environment before ultrasonic treatment. This results in good stability of paeoniflorin, which is beneficial for accurate detection. Compared with other extraction solvents, hydrochloric acid solution has the highest extraction efficiency and requires the fewest processing steps. This invention improves the preparation method of the test sample. After separation and purification, the test sample, when combined with the chromatographic conditions of this invention, yields a stable baseline with minimal interference, and the paeoniflorin chromatographic peaks show good resolution.

[0021] In a further embodiment, the volume ratio of hydrochloric acid to water in the hydrochloric acid solution is 3-7:100;

[0022] Preferably, the volume ratio of hydrochloric acid to water is 5:100.

[0023] The present invention has found through experiments that in hydrochloric acid solutions, the volume ratio of hydrochloric acid to water is 3-7:100, and the extraction effect is not significantly different. However, when the volume ratio of hydrochloric acid to water is controlled at 3:100, for example, adding 3 ml of hydrochloric acid to every 100 ml of water, the solution becomes alkaline after mixing with the sample. Paeoniflorin has poor stability when heated in alkaline conditions. When the volume ratio of hydrochloric acid to water is controlled at 7:100, for example, adding 7 ml of hydrochloric acid to every 100 ml of water, a precipitate forms during sample shaking extraction, requiring filtration to remove the precipitate, which is a complex process and prone to errors. Therefore, as a better approach, the volume ratio of hydrochloric acid to water is controlled at 5:100, for example, adding 5 ml of hydrochloric acid to every 100 ml of water for extraction.

[0024] The hydrochloric acid solution described in this invention is an aqueous solution of concentrated hydrochloric acid (mass fraction of approximately 37%) and water, and the volume of hydrochloric acid refers to the volume of the concentrated hydrochloric acid used.

[0025] In a further embodiment, the mass-to-volume ratio of the contents of the compound Tianqi stomach pain capsule to the hydrochloric acid in the added hydrochloric acid solution is 1:0.5.

[0026] The compound Tianqi stomach pain capsule formula is alkaline. Experiments have shown that 0.5 ml of hydrochloric acid (i.e., concentrated hydrochloric acid used to prepare hydrochloric acid solution) needs to be added to each 1 g of compound Tianqi stomach pain capsule sample to adjust the pH value to neutral, which is beneficial for detection.

[0027] A further proposed approach involves ultrasonic treatment with a power of 400W, a frequency of 40KHz, and an ultrasonic duration of 10-30 minutes.

[0028] Preferably, the ultrasound time is 20 minutes.

[0029] A further step is to extract the extract by shaking with water-saturated n-butanol 3-5 times, and then combine the n-butanol extracts.

[0030] Preferably, the extraction is performed by shaking five times with water-saturated n-butanol.

[0031] The experimental results of this invention show that the shaking extraction effect is 5 times > 4 times > 3 times, therefore, 5 times of shaking extraction is selected as the number of shaking extractions.

[0032] In a further step, in step (1), take an appropriate amount of paeoniflorin reference standard, weigh it accurately, and add methanol to prepare a solution containing 0.2 mg per 1 ml.

[0033] A further formulation involves each Compound Tianqi Stomach Pain Capsule containing 0.5g of the herb, and each capsule contains white peony root extract and paeoniflorin (C). 23 H 28 O 11 The total amount should not be less than 0.10 mg / capsule.

[0034] As a specific implementation method, the quality control method of the Compound Panax notoginseng Stomach Pain Capsules of the present invention includes:

[0035] (1) Preparation of reference solution: Take an appropriate amount of paeoniflorin reference standard, weigh it accurately, and add methanol to prepare a solution containing 0.2 mg per ml.

[0036] (2) Preparation of test solution: Take the contents of compound Tianqi stomach pain capsules, grind them into a fine powder, take about 5g, weigh accurately, place in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH value to neutral (usually 0.5ml of hydrochloric acid needs to be added for every 1g of sample), stopper tightly, weigh, sonicate (power 400W, frequency 40KHz) for 20 minutes, cool, weigh again, make up the lost weight with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, extract 5 times with water-saturated n-butanol, 15ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, and the test solution is obtained.

[0037] (3) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A and 0.2% triethylamine phosphate solution was used as mobile phase B, and gradient elution was performed according to the specifications in the table below; the detection wavelength was 230 nm.

[0038] The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 9,000.

[0039]

[0040] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0041] It should be noted that in this invention, 50ml of hydrochloric acid solution (5→100) means that 5ml of hydrochloric acid is diluted with water to 100ml. The same description in the examples has a similar meaning.

[0042] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0043] 1. The original standard for Compound Tianqi Stomach Pain Capsules has limited testing content. The quality control method for Compound Tianqi Stomach Pain Capsules provided by this invention adds high-performance liquid chromatography quantitative detection of paeoniflorin to the original standard, thereby improving the standard level and making it more conducive to the quality control of Compound Tianqi Stomach Pain Capsules.

[0044] 2. The quality control method of the compound Tianqi stomach pain capsule of the present invention has a simple processing procedure, can effectively separate paeoniflorin, has high specificity, good reproducibility, and low detection limit.

[0045] 3. The quality control method of the compound Tianqi stomach pain capsule of the present invention uses acetonitrile and 0.18-0.22% triethylamine phosphate solution as mobile phase, and the gradient elution procedure with the best effect has been explored and discovered.

[0046] 4. This invention explores an optimized test sample preparation method for detecting paeoniflorin in Compound Tianqi Stomach Pain Capsules. Hydrochloric acid solution is used as the extraction solvent to create a neutral environment before ultrasonic treatment. This results in good stability of paeoniflorin, which is beneficial for accurate detection. After separation and purification of the sample using this method, there is less interference, high specificity, and good reproducibility.

[0047] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0048] The accompanying drawings, as part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0049] Figure 1 The chromatogram is obtained by isogradient washing with acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase;

[0050] Figure 2 The chromatograms are obtained by using acetonitrile-0.1% phosphoric acid solution as the mobile phase and adding gradient washing (Table 2);

[0051] Figure 3The chromatogram is obtained by gradient washing using acetonitrile-0.1% phosphoric acid solution as the mobile phase, according to the procedure in Table 3.

[0052] Figure 4 The chromatogram is obtained by gradient washing using acetonitrile-0.1% phosphoric acid solution as the mobile phase, according to the procedure in Table 4.

[0053] Figure 5 The chromatogram is obtained by gradient washing using acetonitrile-0.1% phosphoric acid solution as the mobile phase, according to the procedure in Table 5.

[0054] Figure 6 The chromatogram is obtained by gradient washing using acetonitrile-0.1% phosphoric acid solution as the mobile phase, according to the procedure in Table 6.

[0055] Figure 7 The chromatogram is obtained by gradient washing using acetonitrile-0.2% triethylamine phosphate as the mobile phase, according to the procedure in Table 7. Figure 8 This is the ultraviolet absorption spectrum of paeoniflorin in section 2.1.2 of this invention;

[0056] Figure 9 This invention presents high-performance liquid chromatograms of paeoniflorin detected using chromatographic columns of different brands; wherein, 1: Shiseido AQ(t R =17.100), 2: Merck column (t R =16.858), 3: Sunfire column (t R =16.807min);

[0057] Figure 10 This is the specific high-performance liquid chromatogram of paeoniflorin in this invention; 1: reference standard (tR = 16.588), 2: 200102 (tR = 16.482), 3: negative sample lacking white peony root, 4: white peony root slices batch 200201 (tR = 16.496);

[0058] Figure 11 This is a linear regression trend chart of paeoniflorin;

[0059] Figure 12 This is a high-performance liquid chromatogram (HPLC) of the paeoniflorin column temperature robustness test of the present invention; 1: original sample (tR = 16.807 min), 2: 25℃ (tR = 17.973 min), 3: 35℃ (tR = 15.588 min).

[0060] Figure 13These are high-performance liquid chromatograms for evaluating the robustness of paeoniflorin mobile phases with different ratios: 1: acetonitrile:triethylamine phosphate 12.5:87.5 (tR = 18.347 min), 2: acetonitrile:triethylamine phosphate 13:87 (tR = 16.807 min), 3: acetonitrile:triethylamine phosphate 13.5:86.5 (tR = 15.299 in);

[0061] Figure 14 These are high-performance liquid chromatograms (HPLC) of the robustness of paeoniflorin mobile phase at different flow rates: 1: 0.9 ml / min (tR = 18.749 min), 2: 1.0 ml / min (tR = 16.807 min), 3: 1.1 ml / min (tR = 15.005 min).

[0062] Figure 15 The high-performance liquid chromatograms for evaluating the robustness of paeoniflorin mobile phases at different acidities are as follows: 1: 0.22% triethylamine phosphate (tR = 16.394 min), 2: 0.2% triethylamine phosphate (tR = 16.807 min), 3: 0.18% triethylamine phosphate (tR = 16.959 min).

[0063] Figure 16 These are high-performance liquid chromatograms (HPLC) of paeoniflorin content from 10 different batches: 1: 200102 (tR = 16.807 min), 2: 200103 (tR = 16.956 min), 3: 200302 (tR = 16.905 min), 4: 200402 (tR = 16.849 min), 5: 200603 (tR = 16.807 min), 6: 200902 (tR = 16.813 min), 7: 201102 (tR = 16.809 min), 8: 210102 (tR = 16.684 min), 9: 210403 (tR = 16.663 min), 10: 210602 (tR = 16.680 min).

[0064] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0065] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0066] The compound Tianqi stomach pain capsules used in this invention are all produced by Guilin Sanjin Pharmaceutical Co., Ltd., batch number 200102.

[0067] Instruments: Waters e2695 high performance liquid chromatograph, Waters 2487 detector, Empower 3 chromatography workstation.

[0068] Reagents: Acetonitrile was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0069] Paeoniflorin reference standard, STD (Standardpharm Co., Ltd.), batch number: 20CDX0177-5L-01

[0070] Example 1

[0071] (1) Preparation of reference solution: Take an appropriate amount of paeoniflorin reference standard, weigh it accurately, and add methanol to prepare a solution containing 0.2 mg per ml.

[0072] (2) Preparation of test solution: Take the contents of compound Tianqi stomach pain capsules, grind them into a fine powder, take about 5g, weigh accurately, place in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH value to neutral (usually 0.5ml of hydrochloric acid needs to be added for every 1g of sample), stopper tightly, weigh, sonicate (power 400W, frequency 40KHz) for 20 minutes, cool, weigh again, make up the lost weight with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, extract 5 times with water-saturated n-butanol, 15ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, and the test solution is obtained.

[0073] (3) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.2% triethylamine phosphate solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the detection wavelength was 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 9000.

[0074] Table 1

[0075]

[0076] (4) Determination method: Accurately pipette 10 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0077] Study of the method in Example 2

[0078] 2.1 Selection of chromatographic conditions:

[0079] 2.1.1 Selection of mobile phase

[0080] Chromatographic system investigation: The effective components of white peony are paeoniflorin and paeoniflorin lactone. Since this variety contains a large amount of alkaline components, the test sample will degrade paeoniflorin when it is heated with water during the processing, resulting in systematic deviation. Therefore, paeoniflorin lactone was selected as the indicator component.

[0081] 2.1.1.1. According to the pharmacopoeia's method for detecting white peony root: use octadecylsilane-bonded silica gel as the filler and acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase; perform isogradient washing and the detection wavelength is 230 nm.

[0082] Preparation of the test sample: Weigh approximately 0.5 g of the sample accurately, add 5 ml of dilute ethanol, weigh again, sonicate (400 W power, 40 kHz frequency) for 30 minutes, cool, replenish the lost weight with dilute ethanol, shake well, filter, and collect the filtrate to obtain the test sample.

[0083] The results showed numerous impurities in the peaks, making baseline separation impossible. These impurities may be due to the sample itself containing many polar impurities similar to paeoniflorin. (See chromatogram below.) Figure 1 .

[0084] 2.1.1.2. After peak elution under the original chromatographic conditions, add a gradient elution procedure to remove excess impurities: use octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the specifications in the table below, with a detection wavelength of 230 nm.

[0085] Table 2

[0086]

[0087] The sample was processed using Method 1. As can be seen from the figure, the impurity peaks are significantly reduced. See the chromatogram below. Figure 2 .

[0088] 2.1.1.3. Due to the presence of a small number of impurity peaks interfering with the baseline of the peak shape, the proportion of aqueous phase was reduced for further subdivision. Octadecylsilane-bonded silica gel was used as the packing material, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was performed according to the specifications in Table 3, 4, or 5, with a detection wavelength of 230 nm.

[0089] Table 3

[0090]

[0091] Table 4

[0092]

[0093] Table 5

[0094]

[0095] The chromatograms obtained according to the gradient elution in Table 3 are shown below. Figure 3 The chromatograms obtained by gradient elution according to Table 4 are shown below. Figure 4 The chromatograms obtained by gradient elution according to Table 5 are shown below. Figure 5 .

[0096] The results showed that baseline separation could not be completely achieved. The reasons for this may be due to low sample content, high impurity levels, and an unsuitable mobile phase.

[0097] 2.1.1.4. Improve the preparation method of the test sample by using octadecylsilane-bonded silica gel as the filler, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the specifications in the table below, with a detection wavelength of 230 nm.

[0098] Table 6

[0099]

[0100] Preparation of the test solution: Take the contents of this product under the "Variation in Content" section, grind it finely, take about 5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH value to neutral (usually 0.5ml of hydrochloric acid needs to be added for every 1g of sample), stopper tightly, weigh it, sonicate (power 400W, frequency 40KHz) for 20 minutes, cool, weigh it again, make up the lost weight with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, extract it 5 times with water-saturated n-butanol, 15ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, and the test solution is ready.

[0101] After separation and purification, the peak signal-to-noise ratio was significantly improved, but baseline stability still needs further improvement. See the chromatogram below. Figure 6 .

[0102] 2.1.1.5. Using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.2% triethylamine phosphate solution as mobile phase B, gradient elution is performed according to the specifications in the table below. The detection wavelength is 230 nm. The preparation method of the test solution is the same as that in 2.1.1.4.

[0103] Table 7

[0104]

[0105] After separation and purification, the chromatographic conditions yielded a stable baseline with minimal interference, and the paeoniflorin peak showed good resolution. See the chromatogram below. Figure 7 .

[0106] Most methods for determining the content of paeoniflorin in related preparations use acetonitrile-water series assays. In this project, we conducted a chromatographic study using acetonitrile-0.2% triethylamine phosphate solution at a detection wavelength of 230 nm. The results showed that the linear gradient in Table 6 was found to be within the conventional C1 range. 18 When the column (250 mm in length, 4.6 mm in inner diameter, and 5 μm in particle size) is used, paeoniflorin can be well separated.

[0107] 2.1.2 Wavelength Selection

[0108] The sample was prepared according to the test sample preparation method in Example 1. A full-wavelength scan was performed on the sample and the paeoniflorin reference standard. Paeoniflorin showed maximum absorption at 230 nm. The chromatogram is shown in the attached figure. Figure 8 .

[0109] 2.1.3 Confirmation of the theoretical number of plates

[0110] Three different brands of chromatographic columns were examined. The theoretical plate number was calculated based on the paeoniflorin peak. The separation of the target components is shown in Table 8, and the chromatograms are attached. Figure 9 Considering the differences between chromatographic columns of different brands, it is proposed that the theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 9000.

[0111] Table 8. Separation of paeoniflorin under different chromatographic columns

[0112]

[0113] 2.2 Study on the preparation of test solution

[0114] 2.2.1 Examination of Extraction Methods

[0115] Take an appropriate amount of the contents of this product from the "Variation in Content" section, grind it finely, take about 5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH to neutral (usually 0.5ml of hydrochloric acid is needed for every 1g of sample), stopper tightly, weigh it, and extract it using two methods: ultrasonic treatment (400W power, 40KHz frequency) for 20 minutes and heating under reflux for 1 hour. After cooling, weigh it again, and make up the weight loss with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, and extract it 5 times with 15ml of water-saturated n-butanol each time. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer it to a 5ml volumetric flask, add methanol to the mark, and shake well. Perform each treatment method in triplicate. Inject into the liquid chromatograph, calculate the results, and the results are shown in Table 9.

[0116] Table 9. Results of different extraction methods

[0117]

[0118] The results showed that ultrasonic extraction had a better extraction effect than thermal reflux extraction, therefore ultrasonic extraction was chosen.

[0119] 2.2.2 Investigation of Extraction Solvents

[0120] Take an appropriate amount of the contents of this product under the "Variation in Content" section, grind it into a fine powder, take about 5g, accurately weigh it, and place it in a stoppered conical flask. Accurately add 50ml each of hydrochloric acid solution (5→100), dilute ethanol hydrochloric acid solution (5→100), and methanol hydrochloric acid solution (5→100). Adjust the pH value to neutral (usually 0.5ml of hydrochloric acid needs to be added for every 1g of sample), stopper tightly, weigh it, and sonicate it (power 400W, frequency 40KHz) for 20 minutes. Let it cool, weigh it again, and make up the weight loss with the corresponding solvent. Mix well, filter, and accurately measure 25ml of the filtrate (of which the dilute ethanol hydrochloric acid and methanol hydrochloric acid extracts are evaporated to dryness, and the residue is dissolved in 25ml of water). Extract it 5 times with water-saturated n-butanol, 15ml each time. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer it to a 5ml volumetric flask, add methanol to the mark, and shake well to obtain the product. Each treatment method was performed in triplicate. The samples were injected into the liquid chromatograph, and the results were calculated. The results are shown in Table 10.

[0121] Table 10 Results of determination using different extraction solvents

[0122]

[0123]

[0124] The results showed that hydrochloric acid solution had the highest extraction efficiency and the fewest processing steps among the three solvents (the main reason for the few processing steps is that only a 5-100 aqueous solution of hydrochloric acid is needed, without the need for methanol or ethanol solution). Therefore, hydrochloric acid solution was chosen for extraction.

[0125] 2.2.3 Investigation on the amount of extraction solvent used

[0126] Take an appropriate amount of the contents of this product under the "Variation in Content" section, grind it finely, take about 5g, accurately weigh it, and place it in a stoppered conical flask. Accurately add 50ml (5→100), 35ml (5→70), and 75ml (5→150) hydrochloric acid solution respectively. Adjust the pH to neutral (usually 0.5ml of hydrochloric acid is needed for every 1g of sample), stopper tightly, weigh, and sonicate (power 400W, frequency 40KHz) for 20 minutes. Cool, weigh again, and make up the weight loss with the corresponding solvent. Mix well, filter, and accurately measure 25ml, 15ml, and 35ml of the filtrate respectively. Extract five times with 15ml of water-saturated n-butanol each time. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, and shake well. Perform each treatment method in triplicate. Inject into the liquid chromatograph, calculate, and the results are shown in Table 11.

[0127] Table 11 Results of determination of different solvent dosages

[0128]

[0129] The results showed no significant differences among the three. However, non-standard pipettes were required for the 35ml and 75ml extracts, which could easily lead to errors. Therefore, 50ml hydrochloric acid solution was used for extraction.

[0130] 2.2.4 Examination of extraction time

[0131] Take an appropriate amount of the contents of this product from the "Variation in Content" section, grind it finely, take about 5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH value to neutral (usually 0.5ml of hydrochloric acid is needed for every 1g of sample), stopper tightly, weigh it, and sonicate it (power 400W, frequency 40KHz) for 10, 20 and 30 minutes respectively, cool it, weigh it again, and make up the weight loss with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, and extract it 5 times with water-saturated n-butanol, 15ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, and the product is obtained. Perform each treatment method in triplicate. Inject into the liquid chromatograph, calculate, and the results are shown in Table 12.

[0132] Table 12 Results of measurements at different extraction times

[0133]

[0134] The results showed no significant differences among the three. Considering that 10 minutes is too short, dynamic equilibrium is difficult to achieve, and the deviation is large, and that the data measured at 20 minutes is more stable (with the smallest RSD% value), 20 minutes was chosen as the extraction time.

[0135] 2.2.5 Investigation into the amount of hydrochloric acid added

[0136] Take an appropriate amount of the contents of this product under the "Variation in Content" section, grind it into a fine powder, take about 5g, accurately weigh it, place it in a stoppered conical flask, and accurately add 50ml of hydrochloric acid solution (5→100), 50ml of hydrochloric acid solution (3→100), and 50ml of hydrochloric acid solution (7→100) respectively. Adjust the pH value to neutral (usually 0.5ml of hydrochloric acid needs to be added for every 1g of sample), stopper tightly, weigh it, sonicate (power 400W, frequency 40KHz) for 20 minutes, cool, weigh it again, make up the lost weight with the corresponding solution, mix well, filter, accurately measure 25ml of the filtrate, and extract it 5 times with water-saturated n-butanol, 15ml each time. (When extracting the sample with 50ml of hydrochloric acid solution (7→100), a large amount of precipitate will be released. Filter to remove the precipitate). Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, and the product is ready. Each treatment method was performed in triplicate. The samples were injected into the liquid chromatograph, and the results were calculated. The results are shown in Table 13.

[0137] Table 13 Results of determination for different amounts of hydrochloric acid

[0138]

[0139] The results showed no significant differences among the three. However, when 3 ml of hydrochloric acid was added to every 100 ml of water, the sample became alkaline. Paeoniflorin showed poor stability when heated in water under alkaline conditions. When 7 ml of hydrochloric acid was added to every 100 ml of water, a precipitate formed during shaking extraction, requiring filtration to remove the precipitate. This process was complex and prone to errors. Therefore, 5 ml of hydrochloric acid was added to every 100 ml of water for extraction.

[0140] 2.2.6 Investigation of the extraction solvent by shaking

[0141] Take an appropriate amount of the contents of this product from the "Variation in Content" section, grind it finely, take about 5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH to neutral (usually 0.5ml of hydrochloric acid is needed for every 1g of sample), stopper tightly, weigh it, sonicate (power 400W, frequency 40KHz) for 20 minutes, cool, weigh it again, replenish the lost weight with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, extract it 5 times with water-saturated n-butanol and ethyl acetate respectively, 15ml each time, combine the water-saturated n-butanol or ethyl acetate extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, and the product is obtained. Perform each treatment method in triplicate. Inject into the liquid chromatograph, calculate, and the results are shown in Table 14.

[0142] Table 14 Results of different shaking extraction solvents

[0143]

[0144]

[0145] The results showed that ethyl acetate was not effective in extracting paeoniflorin, so water-saturated n-butanol was chosen as the shaking extraction solvent.

[0146] 2.2.7 Examination of the number of shaking extractions

[0147] Take an appropriate amount of the contents of this product from the "Variation in Content" section, grind it finely, take about 5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH to neutral (usually 0.5ml of hydrochloric acid is needed for every 1g of sample), stopper tightly, weigh it, sonicate (power 400W, frequency 40KHz) for 20 minutes, cool, weigh it again, replenish the lost weight with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, extract with 15ml of water-saturated n-butanol 3, 4, and 5 times respectively, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, and the product is obtained. Perform each treatment method in triplicate. Inject into the liquid chromatograph, calculate, and the results are shown in Table 15.

[0148] Table 15 Results of different shaking extraction times

[0149]

[0150] The results showed that the shaking extraction effect was better in 5 times than in 4 times, and the results obtained in 5 shaking sessions had relatively small fluctuations. Therefore, 5 shaking extraction sessions were selected as the number of shaking extraction sessions.

[0151] 2.2.8 Investigation of the amount of solvent used in shaking extraction

[0152] Take an appropriate amount of the contents of this product from the "Variation in Content" section, grind it finely, take about 5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH to neutral (usually 0.5ml of hydrochloric acid is needed for every 1g of sample), stopper tightly, weigh it, sonicate (power 400W, frequency 40KHz) for 20 minutes, cool, weigh it again, replenish the lost weight with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, extract it 5 times with water-saturated n-butanol, 10ml, 15ml and 20ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, and the product is ready. Perform each treatment method in triplicate. Inject into the liquid chromatograph, calculate, and the results are shown in Table 16.

[0153] Table 16 Results of different shaking extraction times

[0154]

[0155] The results showed that the content was slightly lower with 10ml, while the extraction efficiency was comparable with 15ml and 20ml. Therefore, 15ml of water-saturated n-butanol was selected as the amount of solvent for shaking extraction.

[0156] In summary, the preparation method for the test solution of the compound Tianqi Weitong capsule for the determination of paeoniflorin content is as follows:

[0157] "Take the contents of this product under the content variation section, grind it into a fine powder, take about 5g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of hydrochloric acid solution (5→100), adjust the pH value to neutral (usually 0.5ml of hydrochloric acid needs to be added for every 1g of sample), stopper tightly, weigh it, sonicate (power 400W, frequency 40KHz) for 20 minutes, cool, weigh it again, make up the lost weight with hydrochloric acid solution (5→100), mix well, filter, accurately measure 25ml of the filtrate, extract it 5 times with water-saturated n-butanol, 15ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, and the product is obtained."

[0158] Example 3 Methodological Validation

[0159] 3.1 Specificity: Negative control trial

[0160] Prepare the negative sample solution according to the above-described sample preparation method. For the processing of white peony root: pulverize the white peony root, pass it through a 40-mesh sieve, accurately weigh approximately 1g, place it in a 50ml volumetric flask, add 35ml of dilute ethanol, sonicate for 30 minutes, cool, add dilute ethanol to the mark, shake well, filter, and collect the filtrate. Inject the reference standard, sample, medicinal material, and negative sample into the liquid chromatograph and analyze according to the chromatographic conditions described in the text. See the attached chromatogram. Figure 10 The results indicate that the paeoniflorin in the test sample is derived from white peony root, with negative results and no interference, demonstrating strong specificity.

[0161] Appendix: Preparation method of negative test sample:

[0162] Preparation of negative samples lacking white peony root: Take one prescription amount of the herb, without white peony root, and extract and prepare it according to the current process of Compound Tianqi Stomach Pain Capsules to obtain a negative sample lacking white peony root.

[0163] 3.2 Linearity Study: Accurately weigh 17.41 mg of paeoniflorin and place it in a 5 ml volumetric flask. Dissolve it in an appropriate amount of methanol and dilute to the mark with methanol. Shake well to prepare a stock solution with a concentration of 3.3183 mg / ml. Then, use this stock solution to prepare a series of solutions with different concentrations. Accurately pipette 10 μl of each of the above solutions and inject them into the liquid chromatograph. Measure and record the peak area of ​​each sample. The data collection results are shown in Table 11. Linear regression calculation was performed with the injection volume as the x-axis and the peak area as the y-axis. The regression trend curve is shown in the figure. Figure 11 The components exhibit a good linear relationship within a certain range. The linear range and regression equation are detailed in Table 18.

[0164] Table 17 Data Table for Examining Linear Relationships

[0165]

[0166] Table 18: Results of Linear Regression Calculation

[0167]

[0168] 3.3 Precision: The sample was processed according to the test solution preparation method, and five consecutive injections were performed. The peak area of ​​paeoniflorin was determined, and the results are shown in Table 19. The calculated RSD of paeoniflorin was 2.11%, indicating that the instrument's detection precision meets the technical requirements for content determination.

[0169] Table 19 Precision test results

[0170]

[0171] 3.4 Sample Stability Study: Samples were prepared according to the assay method described in the main text. They were placed at room temperature and injected into the liquid chromatograph at 0, 1, 2, 3, 4, 7, 12, 20, 25, and 30 hours after sample preparation for analysis. The content was calculated, and the results are shown in Table 20. The results show that the average content of paeoniflorin in the sample was 0.156 mg / capsule, with an RSD of 1.77%, indicating that the sample solution was relatively stable within 30 hours and could meet routine analytical needs.

[0172] Table 20 Results of the stability test of the sample solutions

[0173]

[0174] 3.5 Repeatability: The same sample was processed according to the test solution preparation method, with 6 parallel treatments. The samples were injected into a liquid chromatograph for determination, and the content was calculated. The results are shown in Table 21. The results showed that the average content of paeoniflorin in the test sample was 0.157 mg / capsule, with an RSD of 0.87%, indicating good reproducibility of the method.

[0175] Table 21 Results of Repeatability Tests

[0176]

[0177]

[0178] 3.6 Recovery rate: Approximately 2.5 g of the same batch sample as the repeatability test (batch 200102, content determination result: paeoniflorin 0.157 mg / capsule, average fill weight: 0.4962 g) was accurately weighed and added to 50 ml of hydrochloric acid solution containing paeoniflorin reference standard solution (2.5→100). (16.67 mg of reference standard, purity 95.3%, dissolved in hydrochloric acid aqueous solution and diluted to 10 ml, 4 ml was transferred to a 500 ml volumetric flask, and diluted to the mark with hydrochloric acid aqueous solution, concentration 0.01271 mg / ml). The spiking recovery solution was prepared according to the test sample preparation method. Six parallel solutions were prepared and determined. The recovery rate was calculated using the following formula. The results showed that the spiking recovery rate of paeoniflorin was 94.63% (RSD 1.45%). The determination results are shown in Table 22.

[0179]

[0180] Table 22 Results of the recovery test

[0181]

[0182] The experimental results show that the recovery rate of paeoniflorin in the sample meets the requirements, and the content determination method of the present invention is feasible.

[0183] Example 4 Durability Test

[0184] Take the same sample and process it according to the test solution preparation method, and then perform the determination under different conditions.

[0185] 4.1 Investigation at different column temperatures

[0186] The chromatographic column was set to three different temperatures: 25°C, 30°C, and 35°C. The chromatograms are shown in the attached figure. Figure 12 The results are shown in Table 23. The results indicate that the analysis results are quite consistent when performed within the temperature range of 25–35℃.

[0187] Table 23. Measurement results under different column temperatures.

[0188]

[0189] 4.2 Investigation of different proportions of mobile phase

[0190] The mobile phase ratios were adjusted according to the proportions in Tables 24, 25, and 26 below, and the three batches of samples were analyzed. The chromatograms are attached. Figure 13 The results are shown in Table 27. The results indicate that the determination results are comparable when the overall proportion is adjusted by increasing or decreasing the organic phase by 3.5%.

[0191] Table 24 Proportion A

[0192]

[0193] Table 25 Proportion B

[0194]

[0195] Table 26 Proportion C

[0196]

[0197] Table 27 Results of the investigation of different mobile phase ratios

[0198]

[0199] 4.3 Investigation of different flow velocities

[0200] The analytical conditions were performed at three different flow rates: 0.9, 1.0, and 1.1 ml / min. The chromatograms are shown in the attached figure. Figure 14 The results are shown in Table 28. The results indicate that the analysis results are quite consistent within the flow rate range of 0.9 to 1.1.

[0201] Table 28 Measurement results for different flow velocities

[0202]

[0203] 4.4 Investigation of different acidities

[0204] Three batches of samples were analyzed under three different mobile phase acidity conditions: acetonitrile-0.18% triethylamine phosphate, acetonitrile-0.2% triethylamine phosphate, and acetonitrile-0.22% triethylamine phosphate. The chromatograms are attached. Figure 15 The results are shown in Table 29. The results indicate that the determination was performed with different phosphoric acid concentrations within the range of 0.18% to 0.22% triethylamine phosphate, and the results were comparable.

[0205] Table 29. Measurement results under different acidity conditions

[0206]

[0207] 4.5 Investigation of Chromatographic Columns of Different Brands

[0208] Three batches of samples were analyzed using three columns: Waters Sunfire C18 (5μm 4.6×250mm), Shiseido AQ (5μm 4.6×250mm), and Merck Lichrospher RP-18 (5μm 4.6×250mm). Chromatograms are attached. Figure 9 The results are shown in Table 30. The results indicate that the three brands of chromatographic columns yielded comparable results.

[0209] Table 30 Determination results of chromatographic columns of different brands

[0210]

[0211] In summary, the analytical methods established in this paper can be used to obtain results under different conditions and environments.

[0212] Example 5 Scope

[0213] Take three portions each of the fine powder test sample (2.5 g, 5 g, and 7.5 g). Add 50 ml of hydrochloric acid solution to the 2.5 g sample (2.5→100), 50 ml of hydrochloric acid solution to the 5 g sample (5→100), and 50 ml of hydrochloric acid solution to the 7.5 g sample (7.5→100). Process according to the test sample solution preparation method, and inject into the liquid chromatograph for determination. The results are shown in Table 31.

[0214] Table 31 Results of Paeoniflorin Determination Range

[0215]

[0216] The results showed that the content of paeoniflorin could be accurately determined in the range of 0.0826 mg / capsule to 0.2460 mg / capsule.

[0217] Example 6: Determination results of samples from different batches

[0218] Ten batches of test samples were tested using the method described in Example 1. The chromatograms are shown in the attached figure. Figure 16 The results are shown in Table 32.

[0219] Table 32 Test results of different batches of samples

[0220]

[0221] Example 7: Establishment of Content Limits

[0222] Based on the test results of 10 batches of samples, the average content of the 10 batches of samples was 0.173 mg / capsule, and the lowest content was 0.12 mg / capsule (batch 200402). Adjusting for a 20% reduction from the lowest content, this product is tentatively set to contain paeoniflorin (C) per capsule. 23 H28 O 11 The total dose shall not be less than 0.10 mg / capsule.

[0223] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A detection method of compound Radix Bidentis stomachache capsules, characterized in that, It comprises the following steps: (1) Take paeonol glycoside reference substance to prepare reference substance solution; (2) Prepare test sample solution of compound notopterygium stomachache capsule: take the content of compound notopterygium stomachache capsule, add hydrochloric acid solution to adjust the pH value to neutral, weigh, ultrasonic treatment, cool, re-weigh, supplement the lost weight with hydrochloric acid solution, mix, filter; take the filtrate, extract with water-saturated n-butanol, dry, dissolve the residue with methanol to obtain the test sample solution; (3) Take the reference substance solution and the test sample solution respectively for high performance liquid chromatography detection, the conditions of high performance liquid chromatography include: octadecyl silane bonded silica gel as filler; acetonitrile as mobile phase A, 0.18-0.22% triethylamine phosphate solution as mobile phase B, detection by gradient elution; the detection wavelength of high performance liquid chromatography is 230nm; The conditions of gradient elution include: 。 2. The detection method according to claim 1, characterized in that, The theoretical plate number calculated according to paeonol glycoside peak should not be less than 9000.

3. The method of claim 1, wherein, In step (2), the volume ratio of hydrochloric acid to water in the hydrochloric acid solution is 3-7:

100.

4. The detection method according to claim 3, characterized in that, In step (2), the volume ratio of hydrochloric acid to water is 5:

100.

5. The method of claim 1, wherein In step (2), the mass-volume ratio of the content of compound notopterygium stomachache capsule to hydrochloric acid in the added hydrochloric acid solution is 1:0.

5.

6. The assay of any one of claims 1-5, wherein, In step (2), the ultrasonic treatment power is 400W, the frequency is 40KHz, and the ultrasonic treatment time is 10-30 minutes.

7. The detection method according to claim 6, characterized in that, The ultrasonic treatment time is 20 minutes.

8. The assay of any one of claims 1-5, wherein, In step (2), the water-saturated n-butanol is extracted for 3-5 times, and the n-butanol extract is combined.

9. The detection method according to claim 8, characterized in that, The water-saturated n-butanol is extracted for 5 times.

10. The assay of any one of claims 1-5, wherein, In step (1), take an appropriate amount of paeonol glycoside reference substance, accurately weigh, add methanol to prepare a solution containing 0.2mg per 1ml, and obtain it.

11. The assay of any one of claims 1-5, wherein, Each compound notopterygium stomachache capsule contains 0.5g, and each compound notopterygium stomachache capsule contains not less than 0.10mg of white peony root calculated as paeonol glycoside per particle.

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