Method for determining contents of gentiopicroside and paeoniflorin in Baifeng drink

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, and particularly discloses a method for determining the content of gentiopicroside and paeoniflorin in Baifeng drink, and the method for determining the content of gentiopicroside in a sample comprises the following steps: (1) preparing the sample by using 70% ethanol, and performing ultrasonic treatment for 50 minutes; and (2) carrying out quantitative analysis by using a chromatographic column with octadecylsilane chemically bonded silica filler as a liquid chromatography condition and using a methanol-0. 1% acetic acid solution as a mobile phase at a detection wavelength of 274 nm. The method for measuring the content of paeoniflorin in a sample comprises the following steps: (1) sequentially eluting D101 macroporous adsorption resin with 150mL of water and 20% of ethanol, discarding the eluent, eluting with 100mL of 40% of ethanol, collecting the eluent, evaporating to dryness, and fixing the volume; and (2) carrying out quantitative analysis by using a C18 chromatographic column as a liquid chromatographic condition and using a methanol-0. 1% glacial acetic acid solution as a mobile phase at the detection wavelength of 230nm. The determination method disclosed by the invention is high in specificity, stability, repeatability, intermediate precision, accuracy and the like all accord with regulations, and the durability of chromatographic conditions is relatively good.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis technology, and in particular to a method for determining the content of gentiopicrin and paeoniflorin in Bai Feng Yin (a traditional Chinese medicine formula). Background Technology

[0002] Bai Feng Yin Oral Liquid is included in Volume 15 of the "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China for Traditional Chinese Medicine Preparations" (WS3-B-2867-98). It has the effects of tonifying the liver and kidneys, and replenishing qi and blood. It is often used to treat liver and kidney deficiency, qi and blood deficiency, irregular menstruation in women, metrorrhagia, leukorrhea, and soreness of the lower back and knees. The prescription uses black-boned chicken and Cordyceps sinensis as the principal herbs, combined with 19 other medicinal materials including white peony root, anemarrhena rhizome, gentian root, prepared cyperus rhizome, prepared corydalis rhizome, prepared rehmannia root, achyranthes root, astragalus root, lycium root bark, coptis root, mugwort leaf, fritillaria bulb, rehmannia root, angelica root, poria cocos, artemisia annua, bupleurum root, peony bark, and dried ginger. Through the synergistic effect of multiple components and multiple targets, Bai Feng Yin Oral Liquid has significant efficacy in treating menstrual disorders, dysmenorrhea, and menopausal syndrome caused by deficiency of both qi and blood combined with liver qi stagnation.

[0003] Bai Feng Yin oral liquid has a complex medicinal composition and a complicated preparation process. Furthermore, there are synergistic effects among the various components in the formulation. Therefore, the stability and reproducibility of the active ingredient group are crucial to ensuring clinical efficacy. However, current standards only include amino acid identification, saponin reaction, and thin-layer chromatography identification of Angelica sinensis in the identification section; the content determination section only includes total nitrogen content determination. Therefore, it is difficult to effectively control the production process and product quality of this formulation, failing to meet the relevant requirements for drug quality control and thus hindering its full guarantee of clinical efficacy. Summary of the Invention

[0004] This invention provides a method for determining the content of gentiopicrin and paeoniflorin in Bai Feng Yin, in order to solve the problems mentioned in the background art.

[0005] This invention provides a method for determining the content of gentiopicrin in Bai Feng Yin (a traditional Chinese medicine formula), comprising the following steps:

[0006] (1) Preparation of test solution: Take 10 mL of test solution, place it in a 25 mL volumetric flask, add an appropriate amount of extraction solvent, sonicate, cool, add extraction solvent to make up to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0007] The extraction solvent described in this invention is a commonly used organic solvent in the art, including but not limited to methanol, ethanol, acetonitrile, acetone, ethyl acetate, dichloromethane, and chloroform. In some embodiments, the extraction solvent is methanol, ethanol, or acetonitrile; in some more specific embodiments, the extraction solvent is methanol or ethanol; in some more specific embodiments, the extraction solvent is ethanol with a volume concentration of 10-100%; and in some more specific embodiments, the extraction solvent is ethanol with a volume concentration of 70%.

[0008] The ultrasonic treatment described in this invention is a method for extracting components using the cavitation, mechanical, and thermal effects of ultrasound waves at 20kHz-40kHz. The ultrasonic treatment time is 10-60 minutes. In some embodiments, the ultrasonic frequency is 40kHz, the ultrasonic power is 125-200W, and the extraction time is 20-50 minutes; in some more specific embodiments, the ultrasonic frequency is 40kHz, the ultrasonic power is 200W, and the extraction time is 50 minutes.

[0009] (2) Preparation of reference solution: Take gentiopicroside reference standard, accurately weigh it, add methanol to prepare a solution containing 0.3 mg of gentiopicroside per 1 mL.

[0010] (3) Chromatographic analysis conditions: The chromatographic column is packed with alkyl-bonded silica gel; the mobile phase composition is methanol-0.1% acetic acid solution, and the mobile phase ratio is (5-30):(95-70); the column temperature is 20-40℃, the detection wavelength is 254-320nm; the flow rate is 0.5-1.5mL / min; the theoretical plate number calculated based on the gentiopicroside peak should not be less than 3000.

[0011] The alkyl-bonded silica gel described in this invention is used as a filler, including but not limited to ethane, butyl, heptyl, octyl, cyclohexyl, dodecyl, and octadecyl-bonded silica gels. In some embodiments, octadecyl-bonded silica gel is used as the filler.

[0012] In some embodiments of the chromatographic analysis conditions described in this invention, the mobile phase is methanol-0.1% acetic acid solution (19-20):(81-80); column temperature: 28-32℃; flow rate: 0.9-1.1 mL / min; detection wavelength: 254-274 nm; in some more specific embodiments, the mobile phase is methanol-0.1% acetic acid solution (20:81); column temperature: 30℃; detection wavelength: 274 nm; flow rate: 1.0 mL / min.

[0013] (4) Determination method: Take 5 μL of reference solution and 10 μL of test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0014] This invention also provides a method for determining the paeoniflorin content in Bai Feng Yin, comprising the following steps:

[0015] (1) Preparation of test solution: Accurately measure 10 mL of this product, pass it through a macroporous adsorption resin (column inner diameter 2 cm, column height 10 cm), elute with 150 mL of water, discard the aqueous solution, elute again with the elution solvent, discard the eluent, continue eluting with the elution solvent, collect the eluent, evaporate to dryness, dissolve the residue in solvent, transfer to a 50 mL volumetric flask, add solvent to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0016] The elution solvent and replenishing solvent described in this invention are commonly used organic solvents in the art, including but not limited to methanol and ethanol. In some embodiments, the elution solvent is ethanol with a volume concentration of 10-100%; in some more specific embodiments, the elution solvent is ethanol, and the volume concentration of the elution solvent used in the first elution is 20%; the volume concentration of the elution solvent used in the second elution is 40%. In some embodiments, the replenishing solvent is methanol or ethanol; in some more specific embodiments, the replenishing solvent is methanol with a volume concentration of 10-100%; in some more specific embodiments, the replenishing solvent is methanol with a volume concentration of 100%.

[0017] The sample described in this invention uses a macroporous adsorption resin commonly used in the art, including but not limited to D101 type macroporous adsorption resin, DA201 type macroporous adsorption resin, AB-8 type macroporous adsorption resin, and HPD series macroporous adsorption resin. In some embodiments, the macroporous adsorption resin is D101 type macroporous adsorption resin.

[0018] (2) Preparation of reference solution: Take paeoniflorin reference standard, accurately weigh it, add methanol to prepare a solution containing 60 μg of paeoniflorin per 1 mL.

[0019] (3) Chromatographic conditions: The chromatographic column is packed with alkyl-bonded silica gel, the mobile phase is methanol-0.1% glacial acetic acid solution, and the ratio of the mobile phase is (20-30):(80-70); the column temperature is 20-40℃, the detection wavelength is 230-254nm, the flow rate is 0.5-1.5mL / min, and the theoretical plate number calculated based on the paeoniflorin peak should not be less than 2000.

[0020] The alkyl-bonded silica gel described in this invention is used as a filler, including but not limited to ethane, butyl, heptyl, octyl, cyclohexyl, dodecyl, and octadecyl-bonded silica gels. In some embodiments, octadecyl-bonded silica gel is used as the filler.

[0021] In some embodiments of the chromatographic analysis conditions described in this invention, the mobile phase is methanol-0.1% glacial acetic acid solution (22-29:78-71); column temperature: 28-32℃; flow rate: 0.9-1.1 mL / min; detection wavelength: 230-254 nm; in some more specific embodiments, the mobile phase is methanol-0.1% glacial acetic acid solution (22:78); column temperature: 30℃; detection wavelength: 230 nm; flow rate: 1.0 mL / min.

[0022] (4) Determination method: Take 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0023] The method for determining the content of gentiopicrin and paeoniflorin in Bai Feng Yin provided by this invention has the following beneficial effects:

[0024] (1) Excellent specificity: It can specifically identify and measure gentiopicrin and paeoniflorin in Bai Feng Yin, effectively eliminating interference from other components in the matrix;

[0025] (2) Validation indicators meet the standards: The stability, repeatability, intermediate precision and recovery rate of this method meet the relevant regulations of this field on the validation of methods for the determination of the content of Chinese herbal medicine components, ensuring that the determination results are accurate and reliable;

[0026] (3) Good durability of chromatographic conditions: The liquid chromatography conditions used can still stably achieve effective separation and quantification of target components under slight changes (such as fine adjustment of mobile phase ratio, small fluctuation of column temperature, etc.);

[0027] In summary, the liquid chromatography conditions of this invention can be stably and accurately used for the determination of gentiopicrin and paeoniflorin in Bai Feng Yin, providing reliable technical support for the quality control of Bai Feng Yin. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Chromatogram of gentiopicroside specificity test;

[0030] Figure 2 Linear chromatogram of gentiopicroside;

[0031] Figure 3 Chromatogram of paeoniflorin specificity test;

[0032] Figure 4 This is the linear chromatogram of paeoniflorin. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are also within the scope of protection of the present invention.

[0034] Instruments and reagents: High-performance liquid chromatograph: LC-2050 high-performance liquid chromatography system; Welch Ultimate XB-C18 (4.6×250mm, 5μm) column, ShimNex CSC18 (4.6×250mm, 5μm) column, NanoChormChormCore C18 (4.6×250mm, 5μm) column; Shimadzu AUW220D (0.0001 g) electronic analytical balance; Shimadzu AUW120D (0.0001 g) electronic analytical balance; KQ-250DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0035] Methanol (chromatographic grade, MageRipple (Shanghai) Biotechnology Co., Ltd.); acetic acid, glacial acetic acid, and ethanol were all analytical grade, purchased from Luoyang Haohua Chemical Reagent Co., Ltd.; water was ultrapure water, Grade I water (prepared in the laboratory). Gentianoside reference standard (batch number: 110770-202219, purity: 98.1%) and paeoniflorin reference standard (batch number: 110736-202447, purity: 98.1%) were purchased from the China National Institutes for Food and Drug Control.

[0036] This invention provides a method for determining the content of gentiopicrin in Bai Feng Yin oral preparations, comprising the following steps:

[0037] (1) Selection of chromatographic operating conditions and parameters:

[0038] Chromatographic column: Octadecylsilane-bonded silica gel column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-0.1% acetic acid solution (19-20): (81-80); flow rate: 0.9-1.1 mL / min; column temperature: 28-32℃; detection wavelength: 254-274 nm; theoretical plate number calculated based on gentiopicroside peak should not be less than 3000.

[0039] (2) Preparation of reference solution: Take gentiopicroside reference standard, accurately weigh it, add extraction solvent to prepare a solution containing 0.3 mg of gentiopicroside per 1 mL.

[0040] (3) Preparation of test solution: Take 10 mL of test solution, place it in a 25 mL volumetric flask, add an appropriate amount of extraction solvent, sonicate (frequency 20 kHz-40 kHz, power 125-200 W) for 10-60 min, cool, add extraction solvent to make up to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0041] (4) Determination method: Take 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0042] In some embodiments, in step (1), alkylsilane-bonded silica gel is used as a filler, preferably octadecylsilane-bonded silica gel.

[0043] In some embodiments, high-performance liquid chromatography was performed in accordance with General Chapter 0512 of Part IV of the Chinese Pharmacopoeia 2020.

[0044] In some embodiments, in step (3), the extraction solvent is one of methanol and ethanol. Ethanol is preferred.

[0045] In a more specific embodiment, in step (3), the volume concentration of the ethanol is 10-100%, preferably 70%.

[0046] In some embodiments, in step (3), ultrasonic treatment is used to extract components from the sample. The ultrasonic frequency is 20kHz-40kHz, the ultrasonic power is 125-200W, and the ultrasonic treatment time is 10-60min. Preferably, the ultrasonic frequency is 40kHz, the ultrasonic power is 200W, and the ultrasonic time is 50min.

[0047] In a preferred embodiment of the present invention, the operation method of step (3) is as follows: take 10 mL of the test solution, place it in a 25 mL volumetric flask, add an appropriate amount of 70% ethanol, shake well, sonicate (frequency 40 kHz, power 200 W) for 50 min, cool, add 70% ethanol to make up to the mark, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0048] Example 1: Determination of gentiopicrin content in Bai Feng Yin oral preparation

[0049] This invention provides a method for determining the content of gentiopicrin in Bai Feng Yin oral preparations, comprising the following steps:

[0050] (1) Selection of chromatographic operating conditions and parameters: chromatographic column with octadecylsilane bonded silica gel as packing material; mobile phase of methanol-0.1% acetic acid solution (19:81); flow rate of 1.0 mL / min; column temperature of 30℃; detection wavelength of 274 nm; theoretical plate number calculated based on gentiopicroside peak should not be less than 3000.

[0051] (2) Preparation of reference solution: Take gentiopicrin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.3 mg of gentiopicrin per 1 mL.

[0052] (3) Preparation of test solution: Take 10 mL of test solution, place it in a 25 mL volumetric flask, add 70% ethanol to the mark, shake well, sonicate for 50 min (frequency 40 kHz, power 200 W), filter, and take the filtrate to obtain the test solution.

[0053] (4) Determination method: Take 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0054] Example 2: Determination of gentiopicrin content in Bai Feng Yin oral preparation

[0055] The difference between Example 2 and Example 1 is that in step (1), the mobile phase system and ratio are methanol-0.1% acetic acid solution (20:80), and the rest are the same.

[0056] The measurement results obtained in Examples 1-2 are shown in Table 1:

[0057] Table 1: Chromatographic data for different mobile phases

[0058]

[0059] The results show that the mobile phase ratio used in Example 1 has high separation and theoretical plate number.

[0060] Example 3: Determination of gentiopicrin content in Bai Feng Yin oral preparation

[0061] The difference between this embodiment 3 and embodiment 1 is that in step (1), the wavelength is 254nm, and the rest are the same.

[0062] The results obtained from measurements in Examples 1 and 3 are shown in Table 2:

[0063] Table 2: Influence of different detection wavelengths

[0064] Grouping Detection wavelength (nm) Gentianin content (mg / mL) Example 1 274nm 0.34 Example 3 254nm 0.30

[0065] The results showed that the content of gentiopicrin measured at a detection wavelength of 274 nm was higher than that measured at a wavelength of 254 nm.

[0066] Examples 4-8: Determination of Gentianoside Content in Bai Feng Yin Oral Preparations

[0067] The difference between Examples 4-8 and Example 1 is that different extraction solvents were used to extract the samples in step (3).

[0068] The measurement results obtained in Examples 1 and 4-8 are shown in Table 3:

[0069] Table 3: Results of determination using different extraction solvents

[0070] Grouping Extraction solvent Gentianin content (mg / mL) Example 1 70% ethanol 0.29 Example 4 Anhydrous ethanol 0.28 Example 5 methanol 0.28 Example 6 10% ethanol 0.28 Example 7 30% ethanol 0.28 Example 8 dilute ethanol 0.28

[0071] The results showed that the extraction effects of the two extraction solvents and different concentrations of ethanol were basically the same.

[0072] Examples 9-13: Determination of Gentianoside Content in Bai Feng Yin Oral Preparations

[0073] The difference between this embodiment 9-13 and embodiment 1 is that the ultrasonic treatment time in step (3) is different.

[0074] The measurement results obtained in Examples 1 and 9-13 are shown in Table 4:

[0075] Table 4: Effect of different ultrasonic extraction times

[0076]

[0077]

[0078] The results showed that the content of gentian root extract remained basically consistent within the ultrasonic extraction time range of 10-60 min.

[0079] Examples 14-18: Determination of Gentianoside Content in Bai Feng Yin Oral Preparations

[0080] The difference between Embodiments 14-18 and Embodiment 1 is that in step (3), the ultrasonic processing frequency is 40kHz and the ultrasonic power is different.

[0081] The measurement results obtained in Examples 1 and 14-18 are shown in Table 5:

[0082] Table 5: Effect of different ultrasonic powers on gentiopicrin content

[0083] Grouping Ultrasonic power Gentianin content (mg / mL) Example 1 40kHz, 200W 0.296 Example 14 40kHz, 250W 0.248 Example 15 40kHz, 225W 0.292 Example 16 40kHz, 175W 0.291 Example 17 40kHz, 150W 0.294 Example 18 40kHz, 125W 0.291

[0084] The results showed that different ultrasonic powers had a basically consistent effect on the content of gentiopicrin.

[0085] In summary, Embodiment 1 of the present invention is a preferred embodiment, and the following tests were conducted on its measurement method.

[0086] Methodological Validation

[0087] (1) Specificity test:

[0088] The test solution, the negative sample solution lacking Gentiana macrophylla, and the blank solvent were prepared according to the test solution preparation method in Example 1.

[0089] Accurately pipette the above solution, inject it, and determine the chromatogram according to the chromatographic conditions of Example 1. See [link to example]. Figure 1 (In the figure, S1: negative, S2: test sample, S3: paeoniflorin reference standard).

[0090] Results analysis: Negative samples lacking Gentiana macrophylla and blank solvent chromatography showed no chromatographic peaks at the retention times corresponding to gentiopicrin in the samples, indicating no interference. This demonstrates that the method of the present invention is specific for determining the content of gentiopicrin.

[0091] (2) Examination of linear relationships:

[0092] Accurately weigh gentiopicrin reference standard and dissolve it in methanol to prepare a reference solution containing 0.30 mg / mL. Accurately inject 1 μL, 3 μL, 5 μL, 10 μL, 15 μL, and 20 μL of the reference solution into the liquid chromatograph, respectively, and determine the concentrations under the chromatographic conditions described in Example 1. Plot a standard curve with mass (μg) on ​​the x-axis and peak area on the y-axis. See [link to standard curve]. Figure 2 Table 6 (Linear Relationship of Gentianoside).

[0093] Table 6: Linear Relationship of Gentianoside

[0094] serial number Injection volume (μL) Mass (μg) Peak area 1 1 0.31441 452076 2 3 0.94323 1398730 3 5 1.57205 2339458 4 10 3.14410 4731855 5 15 4.71615 7062173 6 20 6.2882 9382259

[0095] Regression equation: y = 1496753.3211x + 7584.9051, correlation coefficient: r = 1.0000.

[0096] Results analysis: Gentianin showed good linearity in the range of 0.31441-6.2882 μg.

[0097] (3) Precision test:

[0098] S1. Instrument precision test: Take the test sample (batch number: 241002) solution and inject it 6 times consecutively according to the determined determination method. See Table 7 for details.

[0099] Table 7: Precision Test Results (n=6)

[0100]

[0101] Results analysis: With six consecutive injections, the peak area RSD of the test sample was less than 1.5%, indicating that the instrument has good precision.

[0102] S2. Repeatability test:

[0103] Laboratory personnel (A) shall accurately weigh 10 mL of this product (batch number: 241002) (6 portions in total) and perform the procedure according to the established method for preparation and determination of the test solution. See Table 8 for details.

[0104] Table 8: Results of Repeatability Tests (n=6)

[0105]

[0106]

[0107] Results analysis: After six consecutive injections, the RSD of gentiopicroside content in the reference sample was less than 1.5%, indicating that the method has good reproducibility.

[0108] S3. Intermediate precision test:

[0109] Select different determination times, different high-performance liquid chromatographs, and different experimental personnel (B). Take 10 mL of this product (batch number: 241002), accurately weigh it, and perform the operation according to the determined test solution preparation and determination section. The results are shown in Table 9.

[0110] Table 9: Results of Intermediate Precision Test of Gentianoside

[0111]

[0112] Results analysis: The average content of gentiopicroside in the samples measured at different times, using different high-performance liquid chromatographs, and by different experimenters was 0.29 mg / mL, with an RSD of less than 1.5%, indicating good intermediate precision.

[0113] In summary, under the precision conditions (instrument precision, repeatability, and intermediate precision), the RSD of the gentiopicrin content in the samples was less than 1.5%, indicating that the method has good precision.

[0114] (4) Stability test:

[0115] Take 10 mL of Bai Feng Yin sample (batch number: 241002), and operate according to the established method for preparation and determination of the test solution. Inject the sample for determination at 0, 2, 4, 8, 12 and 24 hours after preparation, as detailed in Table 10.

[0116] Table 10: Stability Test Results

[0117]

[0118] Results analysis: The peak area RSD of gentiopicrin was less than 1.5%, and the test solution showed good stability within 24 hours.

[0119] (5) Accuracy test:

[0120] Using the spiking recovery method, accurately weigh 15.00 mg of gentiopicroside reference standard (batch number: 110770-202219, purity: 98.1%) and place it in a 10 mL volumetric flask. Add 70% ethanol to the mark and shake well to obtain a mother liquor reference solution with a concentration of 1.47150 mg / mL.

[0121] Accurately measure 1 mL of the mother liquor reference solution and place it in a 25 mL volumetric flask. Add 70% ethanol to the mark and shake well to obtain a reference solution with a concentration of 0.05886 mg / mL.

[0122] Take 5 mL of this Bai Feng Yin sample (batch number: 241002), accurately measure 6 portions, and place each portion in a 25 mL volumetric flask. Accurately add 1 mL of the above gentianin reference solution to each of the 6 portions, add 70% ethanol to the mark, sonicate for 50 min (frequency 40 Hz, power 200 W), shake well, filter, accurately pipette 10 μL of the filtrate, inject it into the liquid chromatograph, and determine the result. The results are shown in Table 11.

[0123] The content of gentiopicroside in the test sample (batch number: 241002) was 0.2916 mg / mL.

[0124] Table 11: Accuracy Test Results (n=6)

[0125]

[0126] Results analysis: The average recovery rate of gentiopicroside was 98.51%, and the RSD was less than 5.0%, indicating that the method has good accuracy.

[0127] (6) Robustness test: The robustness of different flow rates, column temperatures, mobile phase ratios, chromatographic columns, and acid concentrations to the chromatographic conditions was investigated. The results are shown in Table 12.

[0128] Results analysis: The results obtained by the method of the present invention under different flow rates, column temperatures, mobile phase ratios, acidities, and column conditions are basically consistent, indicating that the method has good robustness.

[0129] Table 12: Results of Durability Tests

[0130]

[0131] In summary, the method for determining the content of gentiopicroside in Bai Feng Yin is highly specific, and its stability, repeatability, intermediate precision, and accuracy all meet the requirements. Furthermore, the chromatographic conditions are robust. Therefore, these liquid chromatography conditions can be used to determine the content of gentiopicroside in Bai Feng Yin.

[0132] This invention also provides a method for determining the content of paeoniflorin in Bai Feng Yin oral preparations, comprising the following steps:

[0133] (1) Chromatographic column: 4.6 mm x 250 mm, 5 μm octadecylsilane bonded silica gel column; mobile phase: methanol-0.1% glacial acetic acid (22-29:78-71); flow rate: 0.9-1.0 mL / min; column temperature: 28-32℃; detection wavelength: 230-254 nm; theoretical plate number calculated based on paeoniflorin peak should not be less than 2000.

[0134] (2) Preparation of reference solution: Take paeoniflorin reference standard, accurately weigh it, add solvent to prepare a solution containing 60 μg of paeoniflorin per mL.

[0135] (3) Preparation of test solution: Accurately measure 10 mL of this product and pass it through a macroporous adsorption resin (column inner diameter 1.5-2 cm, column height 5-15 cm). Elute with 150 mL of water, discard the aqueous solution, elute with 30 mL of elution solvent, discard the eluent, continue eluting with 100 mL of elution solvent, collect the eluent, evaporate to dryness, dissolve the residue in solvent, transfer to a 50 mL volumetric flask, add solvent to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0136] (4) Determination method: Take 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0137] In some embodiments, in step (1), alkylsilane-bonded silica gel is used as a filler, preferably octadecylsilane-bonded silica gel.

[0138] In some of these embodiments, the high-performance liquid chromatography method is referenced to General Chapter 0512 of Part IV of the Chinese Pharmacopoeia 2020 edition.

[0139] In some embodiments, in step (3), the solvent is one of methanol and ethanol. Ethanol is preferred.

[0140] In this more specific embodiment, in step (3), the volume concentration of ethanol is 10-100%, the first elution preferably uses 20% ethanol, and the second elution preferably uses 40% ethanol.

[0141] In some embodiments, in step (3), the processing method is one of different types of macroporous resin elution.

[0142] In this more specific embodiment, in step (3), the resin used for elution is D101 macroporous adsorption resin.

[0143] In a preferred embodiment of the present invention, the operation method of step (3) is as follows: accurately measure 10 mL of the product, pass it through D101 macroporous adsorption resin (column inner diameter 2 cm, column height 10 cm), elute with 150 mL of water, discard the water, elute with 30 mL of 20% ethanol, discard the eluent, continue to elute with 100 mL of 40% ethanol, collect the eluent, evaporate to dryness, dissolve the residue in methanol, transfer to a 50 mL volumetric flask, add methanol to the mark, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0144] Example 19: Determination of paeoniflorin content in Baifengyin oral preparation

[0145] This invention provides a method for determining the content of paeoniflorin in Bai Feng Yin oral preparations, comprising the following steps:

[0146] (1) Selection of chromatographic operating conditions and parameters: chromatographic column: octadecylsilane bonded silica gel as the packing material; methanol-0.1% glacial acetic acid solution (22:78) as the mobile phase; column temperature: 30℃; detection wavelength: 230nm; flow rate: 1.0mL / min; the theoretical plate number calculated based on the paeoniflorin peak should not be less than 2000.

[0147] (2) Preparation of reference solution: Take paeoniflorin reference standard, accurately weigh it, add methanol to prepare a solution containing 60 μg of paeoniflorin per 1 mL.

[0148] (3) Preparation of test solution: Accurately measure 10 mL of this product and pass it through D101 macroporous adsorption resin (column inner diameter 2 cm, column height 10 cm). Elute the sugar with 150 mL of water, discard the aqueous solution, then elute the impurities with 30 mL of 20% ethanol, discard the eluent, and continue to elute with 100 mL of 40% ethanol. Collect the eluent, evaporate to dryness, dissolve the residue in methanol, transfer it to a 50 mL volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0149] (4) Determination method: Take 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0150] Examples 20-22: Determination of paeoniflorin content in Bai Feng Yin oral preparations

[0151] The difference between Examples 20-22 and Example 19 is that different proportions of the mobile phase were selected in step (1).

[0152] The measurement results obtained in Examples 20-22 are shown in Table 13.

[0153] Table 13: Chromatographic data for different mobile phases

[0154]

[0155] The results show that the mobile phase ratio used in Example 19 has high separation and theoretical plate number.

[0156] Examples 23-24: Determination of paeoniflorin content in Bai Feng Yin oral preparations

[0157] The difference between Examples 23-24 and Example 19 is that in step (3), different concentrations of elution solvents are used for sample processing.

[0158] The measurement results obtained in Examples 1 and 23-24 are shown in Table 14:

[0159] Table 14: Chromatographic data for different elution solvents

[0160] Grouping Elution solvent Paeoniflorin content (mg / mL) Example 19 40% ethanol 0.1782 Example 23 30% ethanol 0.1520 Example 24 60% ethanol 0.1517

[0161] The results showed that the elution effect was basically the same for different concentrations of elution solvent used in Example 19.

[0162] Examples 25-27: Determination of paeoniflorin content in Bai Feng Yin oral preparations

[0163] The difference between Examples 25-27 and Example 19 is that in step (3), different types of macroporous adsorption resins are used to treat the samples.

[0164] The results obtained from measurements in Examples 19 and 25-27 are shown in Table 15:

[0165] Table 15: Chromatographic data of samples from different types of macroporous adsorption resins

[0166] Grouping Handling method Paeoniflorin content (mg / mL) Example 1 D101 macroporous adsorption resin elution 0.1769 Example 25 DA201 macroporous adsorption resin elution 0.1640 Example 26 AB-8 type macroporous adsorption resin, 0.1680 Example 27 HPD series macroporous adsorption resins 0.1633

[0167] The results showed that different types of macroporous adsorption resins had basically the same effect on the sample treatment.

[0168] In summary, Embodiment 19 of the present invention is a preferred embodiment, and the following tests were conducted on its measurement method.

[0169] Methodological Validation

[0170] (1) Specificity test:

[0171] The test solution, negative sample solution lacking white peony root and peony bark, and blank solvent were prepared according to the test solution preparation method of Example 19.

[0172] Accurately pipette the above solution, inject it, and determine the chromatogram according to the chromatographic conditions of Example 19. See [link to chromatogram]. Figure 3 (In the figure, S1: negative, S2: test sample, S3: paeoniflorin reference standard).

[0173] Results analysis: In the negative samples lacking white peony root and peony bark, and in the blank solvent chromatogram, there were no chromatographic peaks at the retention times corresponding to paeoniflorin in the samples, indicating no interference. This shows that the method of the present invention is specific for determining the content of paeoniflorin.

[0174] (2) Examination of linear relationships:

[0175] Accurately weigh 6.89 mg of paeoniflorin reference standard, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain a stock solution of reference standard with a concentration of 67.5909 μg / mL. Accurately pipette 1 μL, 3 μL, 5 μL, 7 μL, and 9 μL of the reference standard solution, respectively, into a liquid chromatograph, and determine the concentration under the chromatographic conditions of Example 19. Plot a standard curve with mass (μg) on ​​the x-axis and peak area on the y-axis. See Figure 4 Table 16.

[0176] Table 16: Linear Relationship of Paeoniflorin

[0177] serial number Injection volume (μL) Mass (μg) Peak area 1 1 67.5909 76953 2 3 202.7727 236471 3 5 337.9545 424588 4 7 473.1363 591958 5 9 608.3181 759025

[0178] Regression equation: y = 1272.0877x + 12108.7500

[0179] Correlation coefficient: r = 0.9997

[0180] Results analysis: Paeoniflorin showed good linearity in the range of 67.5909-608.3181 μg.

[0181] (3) Precision test:

[0182] S1. Instrument precision test

[0183] Take the test sample (batch number: 241002) solution and inject it 6 times consecutively according to the determined assay method, as detailed in Table 17.

[0184] Table 17: Precision Test Results (n=6)

[0185]

[0186] Results analysis: With six consecutive injections, the peak area RSD of the test sample was less than 1.5%, indicating that the instrument has good precision.

[0187] S2. Repeatability test:

[0188] Laboratory personnel (A) shall accurately weigh 10 mL of this product (batch number: 241002) (6 portions in total) and perform the procedure according to the established test solution preparation and determination method. See Table 18 for details.

[0189] Table 18: Results of Repeatability Tests (n=6)

[0190]

[0191] Results analysis: After six consecutive injections, the RSD of gentiopicroside content in the reference sample was less than 1.5%, indicating that the method has good reproducibility.

[0192] S3. Intermediate precision test:

[0193] Select different determination times, different high-performance liquid chromatographs, and different experimental personnel (B). Take 10 mL of this product (batch number: 241002), accurately weigh it, and perform the operation according to the determined test solution preparation and determination section. The results are shown in Table 19.

[0194] Table 19: Results of Intermediate Precision Test of Gentianoside

[0195]

[0196] Results analysis: The average content of paeoniflorin in the samples measured at different times, using different high-performance liquid chromatographs, and by different experimenters was 0.17 mg / mL, with an RSD of less than 1.5%, indicating that the method has good intermediate precision.

[0197] In summary, under the precision conditions (instrument precision, repeatability, and intermediate precision), the RSD of the paeoniflorin content in the samples was less than 1.5%, indicating that the method has good precision.

[0198] (4) Stability test:

[0199] Take 10 mL of Bai Feng Yin sample (batch number: 241002), and operate according to the established method for preparation and determination of the test solution. Inject the sample for determination at 0, 2, 4, 8, 12 and 24 hours after preparation, as detailed in Table 20.

[0200] Table 20 Stability Test Results

[0201] Time (h) 0 2 4 8 12 24 average value RSD (%) Peak area 362030 362136 362042 370128 370036 370505 366146 1.2

[0202] Results analysis: The peak area RSD of paeoniflorin was less than 1.5%, and the test solution showed good stability within 24 hours.

[0203] (5) Accuracy test:

[0204] Using the spiking recovery method, 11.04 mg of paeoniflorin reference standard (batch number: 110736-202246, purity: 96.7%) was accurately weighed and placed in a 25 mL volumetric flask. Methanol was added to the mark, and the solution was shaken well to obtain a mother liquor reference solution with a concentration of 0.42703 mg / mL.

[0205] ① Accurately measure 1 mL of the mother liquor reference solution and place it in a 50 mL volumetric flask. Add methanol to make up to the mark and shake well to obtain a reference solution with a concentration of 8.54060 μg / mL.

[0206] ② Accurately measure 2 mL of the mother liquor reference solution, place it in a 50 mL volumetric flask, add methanol to make up to the mark, and shake well to obtain a reference solution with a concentration of 17.18120 μg / mL.

[0207] ③ Accurately measure 3 mL of the mother liquor reference solution, place it in a 50 mL volumetric flask, add methanol to make up to the mark, and shake well to obtain a reference solution with a concentration of 25.62180 μg / mL.

[0208] Take 5 mL of Bai Feng Yin sample (batch number: 241002), accurately measure 3 portions, and pass them separately through D101 macroporous adsorption resin (column inner diameter 2 cm, column height 10 cm). Elute with 150 mL of water, discard the aqueous solution, then elute with 30 mL of 20% ethanol, discard the eluent, and continue eluting with 100 mL of 40% ethanol. Collect the eluent, evaporate to dryness, add 1 mL of 0.42703 mg / mL reference standard stock solution to the residue, dissolve in methanol in a 50 mL volumetric flask, dilute to the mark, shake well, filter, and collect the filtrate to obtain the final product.

[0209] Take 5 mL of Bai Feng Yin sample (batch number: 241002), accurately measure 3 portions, and pass them separately through D101 macroporous adsorption resin (column inner diameter 2 cm, column height 10 cm). Elute with 150 mL of water, discard the aqueous solution, then elute with 30 mL of 20% ethanol, discard the eluent, and continue eluting with 100 mL of 40% ethanol. Collect the eluent, evaporate to dryness, add 2 mL of 0.42703 mg / mL reference standard stock solution to the residue, dissolve in methanol in a 50 mL volumetric flask, dilute to the mark, shake well, filter, and collect the filtrate to obtain the final product.

[0210] Take 5 mL of Bai Feng Yin sample (batch number: 241002), accurately measure 3 portions, and pass each portion through D101 macroporous adsorption resin (column inner diameter 2 cm, column height 10 cm). Elute with 150 mL of water, discard the aqueous solution, then elute with 30 mL of 20% ethanol, discard the eluent, and continue eluting with 100 mL of 40% ethanol. Collect the eluent, evaporate to dryness, add 3 mL of 0.42703 mg / mL reference standard stock solution to the residue, dissolve in methanol in a 50 mL volumetric flask, dilute to the mark, shake well, filter, and collect the filtrate. The results are shown in Table 21.

[0211] The paeoniflorin content in the test sample (batch number: 241002) was 0.1716 mg / mL.

[0212] Table 21: Accuracy Test Results (n=9)

[0213]

[0214] Results analysis: The average recovery rate of paeoniflorin was 98.69%, and the RSD was less than 5.0%, indicating that the method has good accuracy.

[0215] (6) Durability test:

[0216] The robustness of this chromatographic condition to different flow rates, column temperatures, columns, and acid concentrations was investigated, and the results are shown in Table 22.

[0217] Results analysis: The results obtained by the method of the present invention under different flow rates, column temperatures, acidities and column conditions are basically consistent, indicating that the method has good robustness.

[0218] Table 22: Results of Durability Tests

[0219]

[0220]

[0221] In summary, the method for determining the paeoniflorin content in Baifengyin is highly specific, and its stability, repeatability, intermediate precision, and accuracy all meet the requirements. Furthermore, the chromatographic conditions are robust. Therefore, these liquid chromatography conditions can be used for the determination of paeoniflorin content in Baifengyin.

[0222] The above research results show that the method for determining the content of gentiopicrin and paeoniflorin in Baifengyin has strong specificity, and its stability, repeatability, intermediate precision, and accuracy all meet the requirements. In addition, the chromatographic conditions have good robustness. Therefore, these liquid chromatography conditions can be used for the quantitative analysis and determination of gentiopicrin and paeoniflorin in Baifengyin.

[0223] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining the content of gentiopicrin in Bai Feng Yin (a traditional Chinese medicine formula), characterized in that, Includes the following steps: (1) Chromatographic conditions: Alkylsilane-bonded silica gel column as the packing material; The mobile phase is methanol-0.1% phosphoric acid solution, with a methanol-to-0.1% phosphoric acid solution ratio of (5-30):(95-70); column temperature is 20-40℃; detection wavelength is 254-320nm; flow rate is 0.5-1.5mL / min; the theoretical plate number calculated based on the gentiopicroside peak should not be less than 3000. (2) Preparation of reference solution: Take gentiopicroside reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.3-0.5 mg of gentiopicroside per L mL; (3) Preparation of test solution: Take 10 mL of test solution, put it in a 25 mL volumetric flask, add an appropriate amount of extraction solvent, sonicate at 125-250 W and 20-40 kHz for 10-60 min, cool, add extraction solvent to make up to the mark, shake well, filter, and take the filtrate to obtain the test solution. (4) Determination method: Take 5 μL of reference solution and 10 μL of test solution respectively, inject them into the liquid chromatograph, and determine the result.

2. The method for determining the content of gentiopicrin in Bai Feng Yin according to claim 1, characterized in that, The liquid chromatography conditions in step (1) are as follows: using octadecylsilane-bonded silica gel as the packing material; The mobile phase was methanol-0.1% acetic acid solution, with a methanol-to-0.1% phosphoric acid solution ratio of (19-20):(81-80); column temperature: 28-32℃; flow rate: 0.9-1.0 mL / min; detection wavelength: 254-274 nm.

3. The method for determining the content of gentiopicrin in Bai Feng Yin according to claim 1, characterized in that, The extraction solvent in step (3) is either ethanol or methanol.

4. The method for determining the content of gentiopicrin in Bai Feng Yin according to claim 3, characterized in that, The volume concentration of the ethanol is 10-100%.

5. The method for determining the content of gentiopicrin in Bai Feng Yin according to claim 1, characterized in that, In step (3), the ultrasonic treatment frequency is 20-40kHz, the power is 125-200W, and the ultrasonic treatment time is 20-50min.

6. A method for determining the paeoniflorin content in Bai Feng Yin (a traditional Chinese medicine formula), characterized in that, Includes the following steps: (1) Chromatographic conditions: chromatographic column with alkylsilane-bonded silica gel as the packing material; The mobile phase was methanol-0.1% glacial acetic acid solution, with a methanol-to-0.1% glacial acetic acid solution ratio of (20-30):(80-70); the column temperature was set at 20-40℃, the detection wavelength at 230-254nm, and the flow rate at 0.5-1.5mL / min; the theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 2000. (2) Preparation of reference solution: Take paeoniflorin reference standard, accurately weigh it, add methanol to prepare a solution containing 60 μg of paeoniflorin per L mL; (3) Preparation of test solution: Accurately measure 10 mL of this product, treat it by ultrasound or macroporous adsorption resin, transfer it to a 50 mL volumetric flask, add solvent to the mark, shake well, filter, and take the filtrate to obtain the test solution. (4) Determination method: Take 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

7. The method for determining the paeoniflorin content in Bai Feng Yin according to claim 6, characterized in that, In step (1), the chromatographic conditions use octadecylsilane-bonded silica gel as the packing material. The mobile phase was methanol-0.1% glacial acetic acid solution, with a methanol-to-0.1% glacial acetic acid solution ratio of (22-29):(78-71); the column temperature was 28-32℃; the flow rate was 0.9-1.0 mL / min; and the detection wavelength was 230-254 nm.

8. The method for determining the paeoniflorin content in Bai Feng Yin according to claim 6, characterized in that, In step (3), the extraction solvent is 10-100 vol% methanol or 10-100 vol% ethanol; the extraction solvent volume is 50 mL; the sample extraction method is ultrasonic treatment, the extraction power is 125-250 W, the ultrasonic frequency is 20-40 kHz, and the ultrasonic treatment time is 10 min.

9. The method for determining the paeoniflorin content in Bai Feng Yin according to claim 6, characterized in that, In step (3), the macroporous adsorption resin is one of D101 type macroporous adsorption resin and D201 type macroporous adsorption resin; collect 30-100 vol% ethanol or 30-100 vol% methanol eluent.

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