HPLC (High Performance Liquid Chromatography) content determination method of turtle shell decoction solid preparation by quantitative analysis of multi-components by single marker

Abstract

The invention belongs to the technical field of Chinese patent medicine chemical component content detection, and particularly relates to a single-marker multi-component evaluation HPLC content determination method of a turtle shell decoction solid preparation. According to the method, a dilute ethanol heating reflux extraction method is adopted for sample pretreatment, a C18 column is adopted as a chromatographic column, methanol is adopted as a mobile phase A, a phosphoric acid aqueous solution with the concentration of 0.1% is adopted as a mobile phase B, a single wavelength can be adopted through optimization of the method, and meanwhile, baicalin, cinnamaldehyde, paeonol, honokiol, magnolol and chrysophanol in the turtle shell decoction solid preparation are quantitatively detected. The components are not interfered, the accuracy, the precision, the stability, the sample adding recovery rate and the like are good, and the method is suitable for being used as an HPLC content determination method for quantitative analysis of multi-components by single marker of the turtle shell decoction solid preparation and is used for quality detection and control of the turtle shell decoction solid preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical component content detection of traditional Chinese medicines, and particularly relates to a one-test-multiple-evaluation HPLC content determination method for a Biejiajian solid preparation. Background Art

[0002] Biejiajian is composed of 23 Chinese herbs, including Biejiajiao, donkey-hide gelatin, scutellaria, pinellia (processed), codonopsis, dried ginger, magnolia bark (processed with ginger), cinnamon twig, and rhubarb. It has the effects of promoting blood circulation and removing blood stasis, softening and dispersing nodules. This recipe comes from the Synopsis of the Golden Chamber and is commonly used in the clinic to treat liver fibrosis, cirrhosis, angina pectoris, hyperlipidemia, malignant tumors, hepatosplenomegaly, etc., and has achieved good results.

[0003] The commercial preparations of Biejiajian include Biejiajian pills and Biejiajian capsules, both of which are solid preparations with the same prescription and efficacy. The original quality standard of Biejiajian is included in the 1985 edition of the Chinese Pharmacopoeia. Although it has microscopic identification and physical and chemical identification, it lacks content determination items, making it difficult to effectively control its quality.

[0004] Although a few researchers have studied the quantitative detection method of Biejiajian in recent years, the complex composition of this prescription, the mutual interference of complex components, the lack of reference substances, etc., have led to the fact that the currently reported methods can only detect 2 to 3 components in Biejiajian at the same time, and still cannot control its quality well. For example, the HPLC method established by Zhang Qing et al. can detect paeoniflorin and baicalin in Biejiajian by dual wavelength method (Medical Guide 2013: "Determination of paeoniflorin and baicalin in Biejiajian pills by high performance liquid chromatography"); the HPLC method established by Sheng Yunjie et al. can detect paeoniflorin, baicalin and paeonol in Biejiajian by dual wavelength method (Journal of Yunnan University of Traditional Chinese Medicine 2014: "Application of multi-wavelength determination method in the detection of Biejiajian pill extracts").

[0005] The above-mentioned reported methods still have defects such as few substances to be detected simultaneously and the need for dual-wavelength detection. Therefore, it is necessary to develop a one-test-multiple-evaluation method with better detection effect to improve the quality control level of Biejiajian solid preparations. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide a one-test-multiple-evaluation HPLC method for content determination of Biejiajian solid preparation.

[0007] After exploration, the present invention has obtained a one-test-multiple-evaluation HPLC content determination method for Biejiajian solid preparation with better detection effect, and the specific scheme is as follows:

[0008] A one-test-multiple-evaluation HPLC content determination method for a Biejiajian solid preparation comprises the following steps:

[0009] Preparation of reference solution: Using dilute ethanol as solvent, prepare reference solutions of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol and chrysophanol;

[0010] Preparation of test solution: Use dilute ethanol as solvent, grind the test sample into powder, heat and reflux for 30min~45min, make up the lost weight with dilute ethanol, shake well, filter, and take the filtrate as the test solution;

[0011] Injection detection: Take the reference solution and the test solution for injection detection respectively, the detection wavelength is 280nm; the chromatographic column is a C18 column, the column length, inner diameter, and particle size of the chromatographic column are: 250mm, 4.6mm, 5μm respectively; the mobile phase A is methanol, and the mobile phase B is a 0.1% phosphoric acid aqueous solution. The mobile phase elution procedure is as follows:

[0012] .

[0013] The Biejiajian solid preparation is selected from Biejiajian pills or Biejiajian capsules, both of which are reported Biejiajian solid preparations with the same formula.

[0014] Preferably, the reference solution contains 50 μg, 7.5 μg, 15 μg, 15 μg, 10 μg, and 7.5 μg of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol per 1 mL, respectively.

[0015] Preferably, the test solution contains 0.8 g to 1.5 g of the test sample per 50 mL. Further preferably, the test solution contains 0.8 g to 1.3 g of the test sample per 50 mL. Even further preferably, the test solution contains 0.9 g to 1.3 g of the test sample per 50 mL.

[0016] Preferably, the heating reflux temperature during preparation of the test solution is 80°C to 85°C.

[0017] Preferably, the column temperature of the one-measurement-multiple-evaluation HPLC content determination method is 30°C.

[0018] It is well known to those skilled in the art that the term "diluted ethanol" is described in detail in the Pharmacopoeia of the People's Republic of China 2020 edition. Its preparation method and standard requirements are: take 529 mL of ethanol and dilute it to 1000 mL with water. This solution should contain 49.5% to 50.5% (mL / mL) of C2H5OH at 20°C.

[0020] For those skilled in the art, 0.1% phosphoric acid aqueous solution is a commonly used HPLC chromatographic mobile phase, which can be prepared using commercially available phosphoric acid solution (generally with a purity of 85%). It is well known in the art that 0.1% phosphoric acid aqueous solution means 0.1 grams of phosphoric acid dissolved in every 100 mL of water.

[0021] For those of ordinary skill in the art, when the extraction solvent of the test sample is determined, the range of its reflux extraction temperature is constrained by the boiling point of the solvent and the thermal stability of the target substance. Generally speaking, the boiling point of the solvent constrains the lowest temperature of the reflux extraction, and the thermal stability of the target substance constrains the highest temperature of the reflux extraction. Therefore, based on the above basic scientific principles and the basic properties of the substance, those of ordinary skill in the art can reasonably obtain the heating reflux temperature of the test sample solution. For example, the preferred reflux extraction temperature range of the present invention is 80°C to 85°C, that is, the required extraction and quantitative detection requirements can be achieved within this temperature range.

[0022] For those skilled in the art, the concentrations of the reference solution and the test solution can be reasonably selected in combination with the content of the components and the detection efficiency, etc. Obviously, the above-mentioned concentrations of the test solution and the reference solution are only the preferred concentrations used in a specific implementation scheme, and those skilled in the art can reasonably adjust the concentrations of the reference solution and the test solution according to actual experiments.

[0023] The technical solution of the present invention has the following advantages:

[0024] 1. The content determination method of the present invention adopts a simple pretreatment method. A relatively simple extraction method can be used to effectively extract the six components, which is conducive to achieving one measurement and multiple evaluations; and no separate extraction steps are required.

[0025] 2. In the content determination method of the present invention, the HPLC detection mobile phase does not need to use expensive toxic solvents such as acetonitrile, and the solvents used for pretreatment and HPLC elution are commonly used, cheap, and low-toxic methanol and ethanol.

[0026] 3. The content determination method of the present invention can realize the quantitative determination of 6 components at the same time, and there is no interference between the peaks of the 6 components and the peaks of other unknown components in the sample on the detection of the target component. No multi-wavelength detection or separate additional pretreatment procedures are required, and the accuracy, precision, stability, repeatability, sample recovery rate, etc. are all good.

[0027] Obviously, the above advantages of the technical solution of the present invention are achieved by improving the pretreatment method and the HPLC detection conditions including the elution procedure. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A chromatogram of a reference substance for the one-test-multiple-evaluation HPLC content determination method of the present invention;

[0029] Figure 2 The chromatogram of the test sample of the one-test-multiple-evaluation HPLC content determination method of the present invention;

[0030] Figure 3 It is a blank solvent chromatogram of the one-test-multiple-evaluation HPLC content determination method of the present invention;

[0031] Figure 4 This is the chromatogram of the test sample when methanol is used as the extraction solvent;

[0032] Figure 5 This is the chromatogram of the test sample when ethanol is used as the extraction solvent;

[0033] Figure 6 This is the chromatogram of the test product when 50% methanol is used as the extraction solvent;

[0034] Figure 7 This is the chromatogram of the test sample when dilute ethanol is used as the extraction solvent. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments are only used to explain the present invention and are not used to limit the scope of protection of the present invention.

[0036] The sources and models of the instruments, reagents and samples used in the following examples are as follows:

[0037] Instruments: Agilent 1260Ⅱ high performance liquid chromatograph (Agilent Technologies Co., Ltd.), DAD detector, MS105DU electronic balance (Mettler, Switzerland), Model 9960D ultrasonic instrument (CBL Photoelectron Technology Co., Ltd.).

[0038] Test drugs: Baicalin reference substance: batch number 110715-202223, content 97.2%; cinnamaldehyde reference substance: batch number 110710-202223, content 98.8%; paeonol reference substance: batch number 110708-202309, content 99.9%; honokiol reference substance: batch number 110730-201915, content 99.8%; magnolol reference substance: batch number 110729-202316, content 99.2%; chrysophanol reference substance: batch number 110756-201913, content 96.0%; all purchased from China Food and Drug Inspection Institutes.

[0039] The experimental water was ultrapure water, methanol and phosphoric acid were chromatographic grade, and other reagents were analytical grade.

[0040] Biejiajianwan sample: Sinopharm Group Zhonglian Pharmaceutical Co., Ltd.

[0041] Example 1 One-test-multiple-evaluation HPLC content determination method of Biejiajian Pills

[0042] 1. Preparation of test solution: Take the product, grind it into powder, take about 1g, accurately weigh it, put it in a flask, accurately add 50mL of dilute ethanol, weigh it, heat and reflux at 80℃~85℃ for 30min, cool it, weigh it again, make up the lost weight with dilute ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0043] 2. Preparation of reference solution: Take an appropriate amount of reference solution, weigh it accurately, and add dilute ethanol to make a solution containing 50μg, 7.5μg, 15μg, 15μg, 10μg, and 7.5μg of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol per 1mL.

[0044] 3. Sample injection test:

[0045] Take the test solution, reference solution and blank solvent (dilute ethanol) and inject them for detection. Chromatographic conditions:

[0046] The chromatographic column was Shimadzu Shim-pack GIST C18 (250×4.6mm, 5μm); methanol was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, gradient elution was performed according to Table 1, flow rate was 1.0mL / min, detection wavelength was 280nm, column temperature was 30°C. The injection volume was: 20μL sample, 20μL reference, 20μL blank solvent.

[0047] Table 1 Mobile phase elution program

[0048]

[0049] 4. Test results and conclusions

[0050] In the investigation of elution procedures, the pretreatment method and other detection conditions remained unchanged during the preliminary test, and the methodological characteristics such as the chromatographic separation effect of samples with different elution procedures were tested when the same mobile phase A (methanol) and mobile phase B (0.1% phosphoric acid aqueous solution) were used. For example, the content of mobile phase A first increased and then decreased (for example: 40%-95%-40%; 40%-85%-40%, etc.), the content of mobile phase A first decreased and then increased (for example: 50%-10%-50%, etc.), the content of mobile phase A first increased and then decreased and then increased (the initial mobile phase A content of 30% from 0min to 10min, the mobile phase A content of 40% from 11min to 40min, the mobile phase A content of 10% at the 41st minute and maintained for 1min; the mobile phase A content of 30% at the 42nd minute to 45min) and other elution procedures. The chromatogram has many problems, such as the separation degree of baicalin and paeonol cannot meet the requirements of quantitative determination, the separation degree of paeonol and unknown component peaks cannot meet the requirements of quantitative determination, and the separation degree of honokiol and unknown component peaks cannot meet the requirements of quantitative determination. In particular, when other elution procedures are detected, there are unknown peaks that are difficult to separate near the honokiol peak (between the honokiol peak and the honokiol peak; before the honokiol peak, etc.), which cannot meet the requirements of quantitative determination. It was found that the elution procedure in Table 1 can overcome the above problems. Therefore, the elution procedure shown in Table 1 was finally selected.

[0051] Under the test conditions (elution procedure see Table 1), the HPLC chromatograms of the reference substance, test substance and blank solvent are shown in Figure 1 to Figure 3 .like Figure 1 As shown in the figure, the retention times of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol in the reference substance are 7.909min, 10.366min, 15.174min, 28.817min, 31.174min, and 36.118min, respectively. Figure 2 As shown in the figure, the retention times of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol and chrysophanol in the test sample are 7.804min, 10.021min, 15.066min, 28.737min, 31.132min and 36.097min, respectively. Under this method, the chromatographic peaks of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol and chrysophanol in the reference sample and the test sample have good separation, all greater than 1.5, and there is no interference from solvents and other unknown components.

[0052] Example 2 Investigation of extraction solvent

[0053] Take this product, grind it into powder, take about 1g, weigh it accurately, put it in 4 portions, put it in a flask, add methanol, ethanol, 50% methanol, and dilute ethanol 50mL respectively, weigh the weight, heat and reflux for 30min (80℃~85℃), cool, weigh again, make up the lost weight with dilute ethanol, shake well, filter through 0.45μm filter membrane, take 20μL of the filtrate and inject it into liquid chromatograph, detect it under the chromatographic conditions of Example 1, and record the chromatogram.

[0054] The results are shown in Table 2 and Figures 4 to 7 .like Figure 4 As shown in the figure, when methanol is used as the extraction solvent, the retention times of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol are 7.804 min (there is interference from an unknown component peak at the retention time of 6.934 min), 10.036 min, 15.153 min, 28.793 min, 31.177 min, and 36.144 min, respectively. Figure 5 As shown in the figure, when ethanol is used as the extraction solvent, the retention times of baicalin, paeonol, honokiol, magnolol, and chrysophanol are 7.930 min (there is interference from an unknown component peak at the retention time of 7.245 min), 15.542 min, 28.819 min, 31.129 min, and 36.089 min, respectively. Figure 6 As shown in the figure, the retention times of 50% methanol baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol are 7.969min, 10.242min, 15.461min, 28.815min, 31.159min, and 36.099min, respectively. Figure 7 As shown, when dilute ethanol was used as the extraction solvent, the retention times of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol and chrysophanol were 7.804 min, 10.021 min, 15.066 min, 28.737 min, 31.132 min and 36.097 min, respectively.

[0055] The results showed that the peak shape of baicalin extracted by methanol was not good and the extraction was not sufficient; the extraction efficiency of ethanol was not high, and the extraction effects of 50% methanol and dilute ethanol were basically the same. Among them, dilute ethanol was less toxic, economical and environmentally friendly, so dilute ethanol was selected as the extraction solvent.

[0056] Table 2 Extraction solvent investigation results (peak area comparison)

[0057]

[0058] Embodiment 3 Investigation of extraction method and extraction time

[0059] Take this product, grind it into powder, take about 1g, weigh it accurately, put it into 5 portions, put it into flasks, add 50mL of dilute ethanol respectively, weigh it, extract it by ultrasonic (60kHZ; 500W) for 30min, 45min, and 60min respectively, heat it under reflux (80℃~85℃) to extract it for 30min and 45min, let it cool, weigh it again, make up the lost weight with dilute ethanol, shake it well, filter it through 0.45μm filter membrane, take 20μL of the filtrate and inject it into liquid chromatograph, detect it under the chromatographic conditions of Example 1, and record the chromatogram.

[0060] The results are shown in Table 3. The results show that the heating reflux extraction has a better effect, the reflux extraction for 30 minutes is more sufficient, and the content of the components does not increase after 45 minutes of extraction. Therefore, the preferred extraction method is heating reflux for 30 minutes.

[0061] Table 3 Results of investigation on extraction method and extraction time (peak area comparison)

[0062]

[0063] Example 4 Linearity and range investigation

[0064] Take an appropriate amount of the reference solution of Example 1, dilute it step by step into a series of concentrations, and use the chromatographic conditions of Example 1 for detection. Regression is performed with the reference concentration as the abscissa (X) and the peak area as the ordinate (Y), and a standard curve is drawn. The results are shown in Table 4. As can be seen from the results, each component has a good linear relationship within its own range. And the linear range is wide, which is suitable for the quantitative detection of the six components in Biejia Jianwan.

[0065] Table 4 Linear relationship of each component

[0066]

[0067] Example 5 Precision Test

[0068] The reference solution of Example 1 was taken and injected 6 times continuously, and the chromatographic conditions of Example 1 were used for detection. The results are shown in Tables 5 and 6. The retention time RSDs (n=6) of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol were 0.91%, 0.77%, 0.97%, 0.15%, 0.15%, and 0.22%, respectively; the peak area RSDs (n=6) were 0.25%, 1.23%, 0.47%, 0.46%, 0.56%, and 0.86%, respectively, indicating that the method has good precision.

[0069] Table 5 Retention time RSD

[0070]

[0071] Table 6 Peak area RSD

[0072]

[0073] Example 6 Repeatability Test

[0074] According to the method of Example 1, 6 portions of test solution were prepared and injected into liquid chromatograph. The chromatographic conditions of Example 1 were used for detection. The peak area RSDs (n=6) of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol and chrysophanol were calculated. The results were 0.74%, 1.08%, 0.79%, 1.02%, 1.17% and 1.13%, respectively, indicating that the method had good repeatability.

[0075] Example 7 Stability Test

[0076] The test solution of Example 1 was injected into the liquid chromatograph at 0, 2, 4, 6, 8, 12 and 24 h, respectively. The chromatographic conditions of Example 1 were used for detection, and the RSD of the peak area (n=6) was calculated. The results were 0.45%, 1.43%, 0.96%, 1.10%, 1.21% and 1.08%, respectively, indicating that the determination results of this method were stable within 24 h.

[0077] Example 8 Limit of Quantitation and Limit of Detection

[0078] The reference solution of Example 1 was diluted step by step, and the chromatographic conditions of Example 1 were used for sample analysis, and the chromatogram was recorded. The concentration corresponding to the signal-to-noise ratio S / N of 3 was taken as the detection limit, and the concentration corresponding to the S / N of 10 was taken as the quantitative limit. The detection limits of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol were 0.017 μg / ml, 0.030 μg / ml, 0.027 μg / ml, 0.11 μg / ml, 0.11 μg / ml, 0.040 μg / ml, respectively, and the quantitative limits were 0.067 μg / ml, 0.087 μg / ml, 0.067 μg / ml, 0.36 μg / ml, 0.46 μg / ml, and 0.13 μg / ml, respectively. It shows that the detection ability and sensitivity of the method are good, and it is suitable for the quantitative detection of the six components in Biejiajian Pills.

[0079] Example 9: Sample recovery test

[0080] Take this product and grind it into powder, take 6 portions, each of which is about 1g, accurately weigh, and place them in 6 flasks respectively, then accurately add an appropriate amount of the reference solution of Example 1, accurately add 50mL of dilute ethanol, weigh the weight, heat and reflux for 30min, cool, weigh again, make up the lost weight with dilute ethanol, shake well, filter, take 20μl of the filtrate and inject it into the liquid chromatograph, use the chromatographic conditions of Example 1 for detection, determine the peak area of ​​each component, and calculate the recovery rate. The results are shown in Tables 7 and 8. The average sample recovery rates of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol were 98.4%, 96.5%, 97.6%, 96.9%, 97.4%, and 98.1%, respectively, and the RSDs were 1.4%, 1.3%, 1.3%, 1.7%, 1.6%, and 1.5%, respectively, indicating that the method has good accuracy.

[0081] Table 7 Sample recovery test results 1

[0082]

[0083] Table 8 Sample recovery test results 2

[0084]

[0085] Example 10 Sample Determination

[0086] Take 3 batches of samples, prepare the test solution according to the preparation method of Example 1, and use the chromatographic conditions of Example 1 for sample injection analysis. The results are shown in Table 9. In the 3 batches of samples, the content range of each component fluctuates slightly, and the product quality is relatively stable. The content ranges of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol are 3.25-3.74 mg / g, 0.32-0.41 mg / g, 0.58-0.62 mg / g, 0.75-0.83 mg / g, 0.69-0.81 mg / g, and 0.21-0.25 mg / g, respectively.

[0087] Table 9 Sample content determination results

[0088]

Claims

1. A one-test multi-evaluation HPLC method for content determination of Biejiajian solid preparation, wherein the chromatographic column of the one-test multi-evaluation HPLC method is a C18 column, and the column length, inner diameter, and particle size of the chromatographic column are 250 mm, 4.6 mm, and 5 μm, respectively; characterized in that: The one-test-multiple-evaluation HPLC content determination method comprises the following steps: preparation of reference solution: using dilute ethanol as solvent, preparing reference solutions of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol and chrysophanol; Preparation of test solution: Use dilute ethanol as solvent, grind the test sample into powder, heat and reflux for 30min~45min, make up the lost weight with dilute ethanol, shake well, filter, and take the filtrate as the test solution; Injection test: Take the reference solution and the test solution for injection test respectively, the detection wavelength is 280nm, the column temperature is 30℃; the mobile phase A is methanol, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the mobile phase elution procedure is as follows:

2. The one-test multi-analysis HPLC content determination method of a Biejiajian solid preparation according to claim 1, characterized in that: Each 1 mL of the reference solution contains 50 μg, 7.5 μg, 15 μg, 15 μg, 10 μg, and 7.5 μg of baicalin, cinnamaldehyde, paeonol, honokiol, magnolol, and chrysophanol, respectively.

3. The one-test multi-analysis HPLC content determination method of a Biejiajian solid preparation according to claim 1 and 2, characterized in that: The test solution has a concentration of 0.8 g to 1.5 g of the test sample per 50 mL.

4. The one-test multi-analysis HPLC content determination method of a Biejiajian solid preparation according to claim 3, characterized in that: The test solution has a concentration of 0.8 g to 1.3 g of the test sample per 50 mL.

5. The one-test multi-analysis HPLC content determination method of a Biejiajian solid preparation according to claim 4, characterized in that: The test solution has a concentration of 0.9 g to 1.3 g of the test sample per 50 mL.

6. The one-test multi-analysis HPLC content determination method of Biejiajian solid preparation according to claim 1 and 2, characterized in that: The heating reflux temperature of the test solution during preparation is 80°C to 85°C.

7. The one-test multi-analysis HPLC content determination method of Biejiajian solid preparation according to claim 1 and 2, characterized in that: The Biejiajian solid preparation is Biejiajian pills or Biejiajian capsules.

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