Construction method of high performance liquid chromatography for determining contents of elsholtziae herba and application thereof

Abstract

The application provides a construction method for high-performance liquid content determination of Gastrodia elata Blume, and comprises the following steps: precisely sucking control solution and sample solution respectively, and determining by high-performance liquid chromatography, and the control solution is 4-hydroxybenzoic acid solution. The application firstly establishes the high-performance liquid content determination method for the detection of Gastrodia elata Blume, decoction pieces, standard decoction, formula granules and other related preparations; establishes 4-hydroxybenzoic acid as an index component, breaks the defect that there is no index component to reflect the overall content of Gastrodia elata Blume and related preparations, can control the internal quality of Gastrodia elata Blume and related preparations from the overall and macroscopic aspects, guarantees the curative effect of the medicine, and makes the medicine and related preparations have more normal quality control. The method has good stability, high precision, good reproducibility, convenience and easy to master.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of high performance liquid analysis and determination, and particularly relates to a construction method for high performance liquid determination of Lycopodii Herba and application thereof. BACKGROUND

[0002] Lycopodii Herba is a commonly used medicinal material in folk, and is recorded in modern herbal. Lycopodii Herba is the dry aboveground part of Lycopodiastrum casuarinoides (Spring.) Holub. of Lycopodiaceae, has the effects of expelling wind and dampness, relaxing sinews and promoting blood circulation, and is used for treating wind-damp numbness, contusion and sprain, sinew and bone pain, and abdominal distention during menstruation. The medicinal material mainly contains lycopodium alkaloids, terpenes, chromone and aliphatic components, and modern pharmacological studies show that Lycopodii Herba has cholinesterase inhibitory activity, anti-tumor and anti-inflammatory effects. At present, the basic research on Lycopodii Herba is relatively weak, there are a small amount of literatures related to chemical components, and no research on quality control is found, so that the quality of the medicinal material and its preparations is uneven, and the development and effective utilization of Lycopodii Herba are restricted. The present application provides a basis for the quality evaluation and resource development and utilization of Lycopodii Herba and its preparations.

[0003] At present, the existing quality research on Lycopodii Herba and its compound preparations is less, and no related report on high performance liquid analysis and determination is found, so that an effective means for quality control is lacked. SUMMARY

[0004] Therefore, the present application aims to provide a construction method for high performance liquid determination of Lycopodii Herba and application thereof, and aims to construct a high performance liquid determination method for Lycopodii Herba, and provide guidance for the quality control of Lycopodii Herba, medicinal slices and related preparations.

[0005] The present application provides a construction method for high performance liquid determination of Lycopodii Herba, comprising the following steps:

[0006] The control sample solution and the sample solution are precisely taken respectively, and are determined by high performance liquid chromatography, and then the construction method for high performance liquid determination of Lycopodii Herba is obtained.

[0007] The control sample solution is 4-hydroxybenzoic acid solution.

[0008] In the present application, the test solution is a solution of Gout-Relieving Herbs, a solution of Gout-Relieving Herb Decoction Pieces, a solution of Gout-Relieving Herb Standard Decoction or a solution of Gout-Relieving Herb Dispensing Granules. Gout-Relieving Herb is a commonly used medicinal material in folk medicine, which is recorded in many modern herbals. Gout-Relieving Herb is the dry aboveground part of Lycopodiastrum casuarinoides (Spring.) Holub. of Lycopodiaceae, which has the functions of dispelling wind and dampness, relieving the muscles and promoting blood circulation, and is used for treating wind-damp numbness, contusions and sprains, muscle and bone pain, abdominal distension during menstruation and other symptoms. The medicinal material mainly contains lycopodium alkaloids, terpenes, chromones and aliphatic compounds, etc. Modern pharmacological studies have shown that Gout-Relieving Herb has cholinesterase inhibitory activity, anti-tumor and anti-inflammatory effects, etc. At present, the basic research on Gout-Relieving Herb is relatively weak, there are only a few literatures related to chemical components, and there is no research on quality control, which makes it difficult to effectively control the quality of the medicinal material and leads to the long-term existence of the phenomenon that the quality of the medicinal material and its preparations is uneven, thereby restricting the development and effective utilization of Gout-Relieving Herb medicinal resources. The present application provides a basis for the quality evaluation and resource development and utilization of Gout-Relieving Herb medicinal material and its preparations.

[0009] In the present application, the high performance liquid chromatography chromatographic conditions are as follows:

[0010] The chromatographic column is a C18 column.

[0011] The mobile phase A is methanol.

[0012] The mobile phase B is 0.1% acetic acid solution or 0.2% formic acid solution.

[0013] Gradient elution.

[0014] In the present application, the gradient elution is specifically as follows:

[0015] 0-40 min, the mobile phase A is 7-15%, and the mobile phase B is 93-85%.

[0016] In the present application, the 0.1% acetic acid solution is 0.1% acetic acid aqueous solution, and the 0.2% formic acid solution is 0.2% formic acid aqueous solution; both are volume fractions.

[0017] The column temperature of the chromatographic column is 25-35℃. In specific embodiments, the column temperature of the chromatographic column is 25℃, 30℃ or 35℃; when the column temperature is 25℃, 30℃ or 35℃, the chromatogram peak shape and separation effect are better.

[0018] In the present application, the flow rate of the mobile phase is 0.8-1.2 ml / min, and the chromatogram peak shape and separation effect meet the requirements in this flow rate range; the detection wavelength is 260 nm, the injection amount is 8-10 μl, and the theoretical plate number calculated according to the peak of 4-hydroxybenzoic acid should not be less than 5000. In specific embodiments, the flow rate of the mobile phase is 0.8 ml / min, 1.0 ml / min or 1.2 ml / min.

[0019] In the present application, the control solution is prepared according to the following method:

[0020] In the present application, the injection concentration of the 4-hydroxybenzoic acid solution is 1.0072-50.3600 μg / ml, preferably 5-40 μg / ml, and more preferably 8-15 μg / ml; in a specific embodiment, the injection concentration of the 4-hydroxybenzoic acid solution is 10 μg / ml.

[0021] The 4-hydroxybenzoic acid control sample is dissolved in 70% ethanol to obtain a 4-hydroxybenzoic acid solution.

[0022] In the present application, the test solution is prepared according to the following method:

[0023] The test sample and 70% ethanol are mixed, weighed, refluxed or ultrasonically treated, cooled, weighed again, the lost weight is made up with 70% ethanol, shaken, filtered, and the filtrate is obtained;

[0024] In the present application, the chromatogram baseline of 70% ethanol as the extraction solvent is flat, the peak shape of 4-hydroxybenzoic acid is good, and the integration is easier to separate from the baseline, so the solution of 70% ethanol is selected as the extraction solvent for the content determination of Shujincao Formula Granules.

[0025] If the test sample is Shujincao Standard Decoction or Shujincao Formula Granules, the mass of the test sample and the volume of 70% ethanol are (0.05-1.00) g:20 mL; in a specific embodiment, the mass of the test sample and the volume of 70% ethanol are 0.2 g:20 mL; when the sample weight is 0.2 g, the peak shape of the chromatogram is better, the peak area is moderate, and the extraction efficiency is higher.

[0026] If the test sample is Shujincao medicinal material or Shujincao decoction pieces, the mass of the test sample and the volume of 70% ethanol are (0.2-2.0) g:25 mL; in a specific embodiment, the mass of the test sample and the volume of 70% ethanol are 1.0 g:25 mL.

[0027] There is no significant difference between the reflux extraction efficiency and the ultrasonic extraction efficiency, but the ultrasonic extraction method is simple to operate, so the present application preferably uses ultrasonic extraction. In the present application, the power of ultrasonic treatment is 580-620 W, preferably 590-610 W; the frequency of ultrasonic treatment is 35-45 kHz, preferably 38-42 kHz; and the time of ultrasonic treatment is 25-35 min, preferably 28-32 min. In a specific embodiment, the power of ultrasonic treatment is 600 W, the frequency of ultrasonic treatment is 40 kHz, and the time of ultrasonic treatment is 30 min.

[0028] The present application also provides a method for identifying Shujincao, which uses the above technical solution to detect and analyze the detection results.

[0029] The present application firstly establishes a high performance liquid content determination method for the detection of Shujincao medicinal materials, decoction pieces, standard decoction, formula granules and other related preparations. 4-hydroxybenzoic acid is established as an index component, breaking the defect that Shujincao and its related preparations have no index component to reflect the overall content, so that the internal quality of Shujincao and its related preparations can be controlled from the whole and macroscopic, and the efficacy of the drug is ensured, so that the medicinal materials and their related preparations have obtained more normal quality control. The method of the present application has good stability, high precision, good reproducibility, convenience and easy to master. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a mobile phase selection diagram;

[0031] Figure 2 is a 4-hydroxybenzoic acid-contrast ultraviolet absorption spectrum diagram;

[0032] Figure 3 is a 4-hydroxybenzoic acid-test product ultraviolet absorption spectrum diagram;

[0033] Figure 4 is a flow rate investigation result;

[0034] Figure 5 is a column temperature investigation result;

[0035] Figure 6 is an extraction solvent investigation chromatogram;

[0036] Figure 7 is Shujincao formula granule specificity investigation;

[0037] Figure 8 is a 4-hydroxybenzoic acid standard curve diagram. DETAILED DESCRIPTION

[0038] In order to further illustrate the present application, the method for constructing Shujincao high performance liquid content determination and its application provided by the present application are described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.

[0039] Example 1

[0040] 1. Instruments and reagents

[0041] 1.1 Experimental instruments

[0042] Liquid chromatograph: Waters H-Class Plus type ultra-high performance liquid chromatograph;

[0043] Electronic balance: ME204E, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0044] Ultrapure water machine: Cell type 1810A (Shanghai Molier Scientific Instrument Co., Ltd.);

[0045] Ultrasonic cleaner: KQ-600DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0046] 1.2 Reagents and reagents

[0047] Shujincuo control medicinal materials (Shanghai Hongyong Biological Technology Co., Ltd., batch number: 380003-202104);

[0048] 4-hydroxybenzoic acid (China Food and Drug Inspection Research Institute, batch number 101149-202204, purity 100%);

[0049] Methanol, acetic acid, formic acid are chromatographically pure; water is ultrapure water; other reagents are analytical pure;

[0050] Shujincuo medicinal materials batch number: SJC-YC-01, SJC-YC-01, SJC-YC-02, SJC-YC-03, SJC-YC-04, SJC-YC-05, SJC-YC-06, SJC-YC-07, SJC-YC-08, SJC-YC-09, SJC-YC-10, SJC-YC-11, SJC-YC-12, SJC-YC-13, SJC-YC-14, SJC-YC-15, SJC-YC-16, SJC-YC-17, SJC-YC-18, SJC-YC-19, SJC-YC-20;

[0051] Shujincuo decoction pieces batch number: SJC-YP-01, SJC-YP-02, SJC-YP-03, SJC-YP-04, SJC-YP-05, SJC-YP-06, SJC-YP-07, SJC-YP-08, SJC-YP-09, SJC-YP-10, SJC-YP-11, SJC-YP-12, SJC-YP-13, SJC-YP-14, SJC-YP-15, SJC-YP-16, SJC-YP-17, SJC-YP-18, SJC-YP-19, SJC-YP-20;

[0052] Shujincao Standard Decoction Batch No.: SJC-BT-01, SJC-BT-02, SJC-BT-03, SJC-BT-04, SJC-BT-05, SJC-BT-06, SJC-BT-07, SJC-BT-08, SJC-BT-09, SJC-BT-10, SJC-BT-11, SJC-BT-12, SJC-BT-13, SJC-BT-14, SJC-BT-15, SJC-BT-16, SJC-BT-17, SJC-BT-18, SJC-BT-19, SJC-BT-20;

[0053] Shujincao Dispensing Granules Batch No.: SJC-CP-01, SJC-CP-02, SJC-CP-03.

[0054] 2. Content Determination Method Establishment

[0055] 2.1 Chromatographic Conditions and System Suitability Test

[0056] The chromatographic conditions and system suitability test used octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, 0.2% formic acid solution as mobile phase B, gradient elution according to the provisions in the following table; detection wavelength 260 nm. The theoretical plate number calculated according to the 4-hydroxybenzoic acid peak should not be less than 5000.

[0057]

[0058] 2.2 Preparation of Reference Solution

[0059] Take an appropriate amount of 4-hydroxybenzoic acid reference substance, accurately weigh, prepare a solution containing 4-hydroxybenzoic acid 10 μg per 1 ml with 70% methanol as the reference solution.

[0060] 2.3 Preparation of Test Solution

[0061] Take an appropriate amount of medicinal material test solution (pass through No. 3 sieve), take about 1.0 g, accurately weigh, place in a stoppered conical flask, accurately add 70% ethanol 25 ml, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 70% ethanol, shake well, filter, take the filtrate, and obtain.

[0062] Take an appropriate amount of medicinal material test solution (pass through No. 3 sieve), take about 1.0 g, accurately weigh, place in a stoppered conical flask, accurately add 70% ethanol 25 ml, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 70% ethanol, shake well, filter, take the filtrate, and obtain.

[0063] For the standard decoction test solution, accurately weigh about 0.2 g of this product and place it in a stoppered conical flask. Accurately add 20 ml of 70% methanol, weigh the solution, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh the solution again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate.

[0064] For the test solution of the granules, take an appropriate amount of this product, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 20ml of 70% methanol, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0065] 2.4 Determination Method

[0066] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0067] 2.5 Investigation of chromatographic conditions

[0068] 2.5.1 Selection of mobile phase

[0069] Based on the proposed experiment, methanol-0.1% acetic acid solution and methanol-0.2% formic acid solution were selected as the mobile phases for determining the content of *Shujincao* (a traditional Chinese medicine) formula granules. The elution effect was investigated. Figure 1 And Table 1:

[0070] Table 1. Analysis results of different mobile phases

[0071]

[0072] The results showed that when the chromatographic conditions were methanol-0.1% acetic acid solution and methanol-0.2% formic acid solution as the mobile phase, the theoretical plate number and resolution of the 4-hydroxybenzoic acid peak were both high, and both could be used as the mobile phase for detection.

[0073] 2.5.2 Wavelength Selection

[0074] Based on the above-specified experimental conditions, a diode array detector was used to perform full-band scanning of the 4-hydroxybenzoic acid reference solution and the test solution, respectively. See... Figure 2 and Figure 3 .

[0075] 2.5.3 Flow velocity assessment

[0076] Under the proposed experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. (See...) Figure 4 and Table 2;

[0077] Table 2 Analysis results of different flow rates

[0078]

[0079] The results show that when the flow rate is 0.8 ml / min, 1.0 ml / min, 1.2 ml / min, the chromatogram peak shape and separation effect meet the requirements, and the flow rate is determined as 1.0 ml / min.

[0080] 2.5.4 Column temperature investigation

[0081] On the basis of the above determined test conditions, the column temperature of 25°C, 30°C, 35°C was investigated, respectively, as shown in Table 2 and Table 3: Figure 5

[0082] Table 3 Analysis results of different column temperatures

[0083]

[0084] The results show that when the column temperature is 25°C, 30°C, 35°C, the chromatogram peak shape and separation effect meet the requirements, and the detection column temperature is determined as 30°C.

[0085] 2.5.5 Chromatographic conditions and system suitability test results

[0086] The chromatographic conditions and system suitability test use octadecylsilane bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); methanol is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B, which is gradient eluted according to the following table; the detection wavelength is 260 nm. The theoretical plate number calculated by 4-hydroxybenzoic acid peak should not be less than 5000.

[0087]

[0088] 2.6 Investigation of test solution preparation

[0089] 2.6.1 Investigation of extraction method

[0090] Take the product (batch number: SJC-CP-01) in an appropriate amount, grind finely, take about 0.3 g, accurately weigh, put into a conical flask with a plug, accurately add 70% methanol 20 ml, weigh, respectively, use heating reflux, ultrasonic (power 600 W, frequency 40 kHz) method to extract for 30 minutes, cool, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and get it. The results are shown in Table 4.

[0091] Table 4 Investigation results of extraction method

[0092] ​

[0093] From the above, there is no significant difference between reflux and ultrasonic extraction efficiency, so the ultrasonic method with simple operation is selected for extraction.

[0094] 2.6.2 Sample weight investigation

[0095] Take the product (batch number: SJC-CP-01) in an appropriate amount, grind finely, and take about 0.2 g, 0.3 g, and 0.5 g, accurately weigh, place in a conical flask with a stopper, respectively, accurately add 20 ml of 70% methanol, weigh, ultrasonic extraction (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is obtained. The results are shown in Table 5.

[0096] Table 5 Content results of sample weight investigation

[0097]

[0098] The results show that when the sample weight is 0.2 g, the chromatogram peak shape is good, the peak area is moderate, and the extraction efficiency is high. Therefore, the sample weight is determined to be 0.2 g.

[0099] 2.6.3 Extraction time investigation

[0100] Take the product (batch number: SJC-CP-01) in an appropriate amount, grind finely, and take about 0.2 g, accurately weigh, place in a conical flask with a stopper, accurately add 20 ml of 70% methanol, weigh, ultrasonic extraction (power 600 W, frequency 40 kHz) for 20 minutes, 30 minutes, and 40 minutes, respectively, cool, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is obtained. The results are shown in Table 6.

[0101] Table 6 Results of extraction time investigation

[0102]

[0103]

[0104] From the above, 30 minutes of extraction can be sufficient for extraction, so the extraction time of the test sample is determined to be 30 minutes.

[0105] 2.6.4 Extraction solvent investigation

[0106] Take the product (batch number: SJC-CP-01) in an appropriate amount, grind finely, and take about 0.2 g, accurately weigh, place in a conical flask with a stopper, respectively, accurately add water, ethanol, methanol, 30% ethanol, 30% methanol, 70% ethanol, and 70% methanol each 20 ml, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, and take the filtrate, which is obtained. The results are shown in Table 7.Figure 6 and Table 7:

[0107] Table 7 Extraction solvent investigation results

[0108]

[0109] From the above, the extraction efficiency of 70% methanol and 70% ethanol solution is the highest. Since the chromatogram baseline is flat, the peak shape of 4-hydroxybenzoic acid is good, and the integration is easier to separate from the baseline when 70% ethanol is used as the extraction solvent, therefore, the solution of 70% ethanol is selected as the extraction solvent for the content determination of Shujincao Formula Granules.

[0110] In summary, the preparation method of the test sample for the content determination of Shujincao Formula Granules is as follows: take an appropriate amount of the product, grind it finely, take about 0.2 g, accurately weigh it, place it in a conical flask with a plug, accurately add 20 ml of 70% ethanol, weigh it, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 70% ethanol, shake it evenly, filter it, take the filtrate, and it is obtained.

[0111] 2.6.5 Method for the content determination of Shujincao Formula Granules

[0112] The chromatographic conditions and system suitability test use octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); use methanol as the mobile phase A, and 0.2% formic acid solution as the mobile phase B, perform gradient elution according to the following table; the detection wavelength is 260 nm. The theoretical plate number calculated according to the peak of 4-hydroxybenzoic acid should not be less than 5000.

[0113]

[0114] Prepare the reference solution by accurately weighing an appropriate amount of 4-hydroxybenzoic acid reference substance, adding 70% ethanol to prepare a solution containing 10 μg of 4-hydroxybenzoic acid per 1 ml, and it is obtained.

[0115] Prepare the test solution by accurately weighing an appropriate amount of the product, grinding it finely, taking about 0.2 g, accurately weighing it, placing it in a conical flask with a plug, accurately adding 20 ml of 70% ethanol, weighing it, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cooling it, weighing it again, making up the weight loss with 70% ethanol, shaking it evenly, filtering it, taking the filtrate, and it is obtained.

[0116] Prepare the test solution by accurately weighing an appropriate amount of the product, grinding it finely, taking about 0.2 g, accurately weighing it, placing it in a conical flask with a plug, accurately adding 20 ml of 70% ethanol, weighing it, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cooling it, weighing it again, making up the weight loss with 70% ethanol, shaking it evenly, filtering it, taking the filtrate, and it is obtained.

[0117] 2.7 Methodology investigation

[0118] 2.7.1 Investigation of specificity

[0119] Preparation of reference solution: Weigh an appropriate amount of 4-hydroxybenzoic acid reference substance accurately, dissolve it in 70% ethanol to prepare a solution containing 10 μg of 4-hydroxybenzoic acid per 1 ml as the reference solution.

[0120] Preparation of test solution: According to the experimental conditions in "Tentative preparation method of test solution for content determination of Shujincao formula granules" in 2.6.4, prepare the test solution of Shujincao formula granules.

[0121] Preparation of negative control solution: According to the above-determined experimental conditions, prepare a negative control solution without Shujincao formula granules. The results are shown in Figure 7 , from Figure 7 it can be seen that the chromatogram of the negative solution has no interference on the determination of the target peak, indicating that the method has good specificity.

[0122] 2.7.2 Precision investigation

[0123] Inject the 4-hydroxybenzoic acid reference solution continuously for 6 times, record the peak areas, and calculate the RSD value. The results are shown in Table 8.

[0124] Table 8 Results of precision investigation

[0125]

[0126] From the above, it can be seen that the RSD value of the peak area of 4-hydroxybenzoic acid in the precision investigation is 0.1%, indicating good precision of the instrument.

[0127] 2.7.3 Linear relationship

[0128] Weigh an appropriate amount of 4-hydroxybenzoic acid, place it in a 50 mL volumetric flask, dissolve it with 70% ethanol to prepare a solution containing 50.3600 μg / mL (purity 100.0%) of 4-hydroxybenzoic acid, and then dilute it to concentrations of 1.0072 μg / ml, 2.0144 μg / ml, 5.0360 μg / ml, 10.0720 μg / ml, and 25.1800 μg / ml. Inject 10 μl of each solution into the liquid chromatograph for determination. Plot the response curve with the concentration (X, μg / mL) as the abscissa and the peak area (Y) as the ordinate. The results are shown in Table 9 and Figure 8 :

[0129] Table 9 Analysis results of the standard curve of 4-hydroxybenzoic acid

[0130]

[0131] From Figure 8 it can be seen that when the injection concentration of 4-hydroxybenzoic acid is within 1.0072 - 50.3600 μg / ml, the linear relationship is y = 54.1463x - 9.3959, R Figure 8 , = 0.9998, indicating that the 4-hydroxybenzoic acid sample concentration showed a good linear relationship at 1.0072-50.3600 μg / ml.

[0132] 2.7.4 repeatability

[0133] About 0.2 g of the product (batch number: SJC-CP-01) was precisely weighed in six portions, and a test solution was prepared according to the proposed experimental method by the same operator. The 4-hydroxybenzoic acid content of the six test solutions was determined and calculated, and the results are shown in Table 10.

[0134] Table 10 Repeatability test results

[0135]

[0136]

[0137] As can be seen from the table, the RSD value of the 4-hydroxybenzoic acid content obtained from the six repeatability tests was 0.4%, and the method was good in repeatability.

[0138] 2.7.5 intermediate precision

[0139] Two test solutions were prepared according to the proposed method by different personnel (A1, A2) at different times (T1, T2), and were determined on different high-performance liquid chromatography instruments (C1, C2, C3). The content of 4-hydroxybenzoic acid was calculated, and the results are shown in Table 11.

[0140] Table 11 Results of different personnel and time investigation

[0141]

[0142] As can be seen from the above, the RSD value of the 4-hydroxybenzoic acid content determination results was 1.57% when different personnel performed the test at different times, and the method was good in intermediate precision.

[0143] 2.7.6 sample recovery rate

[0144] About 0.1 g of test sample with known content (batch number: SJC-CP-01, 4-hydroxybenzoic acid content 0.75 mg / g) was precisely weighed in six portions, and a certain amount of 4-hydroxybenzoic acid reference solution (concentration 5.174 μg / ml) was precisely added. The test solution was prepared according to the proposed method, and the test solution was injected into the liquid chromatograph for determination, and the recovery rate was calculated, and the results are shown in Table 12. The calculation formula is as follows:

[0145]

[0146] Table 12 Sample recovery test results

[0147]

[0148]

[0149] From the above, the average recovery of 4-hydroxybenzoic acid is 101.0%, and the accuracy of the method is good.

[0150] 2.7.7 Durability Investigation

[0151] 2.7.7.1 Column Durability Investigation

[0152] The specification of the present application was used, and three different types of C18 chromatographic columns (column 1: TC-C18 (250x4.6mm, 5μm), column 2: Shim-pack GIST C18 (250x4.6mm, 5μm), column 3: InertSustain C18 (250x4.6mm, 5μm)) were used to detect the same test sample, and the results are shown in Table 13.

[0153] Table 13 Column Durability Investigation Results

[0154]

[0155] The results show that the RSD value of the content determination results of 4-hydroxybenzoic acid is 1.35% when different types of chromatographic columns are used for detection, and the column durability of the method is good.

[0156] 2.7.8 Stability Experiment

[0157] According to the above proposed experimental conditions, a test sample solution was prepared, and the peak area of 4-hydroxybenzoic acid was measured at 0h, 5h, 10h, 15h, 20h, 24h, respectively. The results are shown in Table 14.

[0158] Table 14 Stability Experiment Results

[0159]

[0160] From the above, the RSD value of the peak area of 4-hydroxybenzoic acid in the sample solution within 24h is 0.5%, which indicates that the test sample solution has good stability within 24 hours.

[0161] 2.8 Sample Content Determination Verification and Method Establishment

[0162] 2.8.1 Medicinal Material Verification

[0163] Take 20 batches of Gout-Relieving Herbs as test samples, prepare test sample solutions according to the method for preparing test sample solutions of medicinal materials in the section of “Preparation of Test Sample Solutions” in 2.3, and determine the peak area, calculate the content of 4-hydroxybenzoic acid, and the results are shown in Table 15.

[0164] Table 15 Content determination results of 20 batches of Gout-Relieving Herbs

[0165]

[0166] From the above, it can be seen that the content determination method can effectively detect Gout-Relieving Herbs.

[0167] 2.8.1 Verification of Decoction Pieces

[0168] Take 20 batches of Gout-Relieving Herbs as test samples, prepare test sample solutions according to the method for preparing test sample solutions of medicinal materials in the section of “Preparation of Test Sample Solutions” in 2.3, and determine the peak area, calculate the content of 4-hydroxybenzoic acid, and the results are shown in Table 16.

[0169] Table 16 Detection results of 20 batches of Gout-Relieving Herbs

[0170]

[0171]

[0172] From the above, it can be seen that the content determination method can effectively detect Gout-Relieving Herbs.

[0173] 2.8.1 Verification of Standard Decoction

[0174] Take 20 batches of Gout-Relieving Herbs as test samples, prepare test sample solutions according to the method for preparing test sample solutions of medicinal materials in the section of “Preparation of Test Sample Solutions” in 2.3, and determine the peak area, calculate the content of 4-hydroxybenzoic acid, and the results are shown in Table 17.

[0175] Table 17 Detection results of 20 batches of Gout-Relieving Herbs

[0176]

[0177]

[0178] From the above, it can be seen that the content determination method can effectively detect Gout-Relieving Herbs.

[0179] 2.7.9 Verification of Formula Granules

[0180] Take 3 batches of Shujincao formula granules as the test product, prepare the test product solution according to the prepared formula granule test product solution method, record the peak area, calculate the content of 4-hydroxybenzoic acid, and the results are shown in Table 18.

[0181] Table 18 Content determination results of three batches of Shujincao formula granules

[0182]

[0183] From the above, it can be seen that the content determination method can effectively detect Shujincao formula granules.

[0184] From the above examples, the present application aims to construct a Shujincao high-performance liquid content determination method, and provide guidance for the quality control of Shujincao medicinal materials, decoction pieces and related preparations. The present application first establishes a high-performance liquid content determination method for the detection of Shujincao medicinal materials, decoction pieces, standard decoction, formula granules and other related preparations. 4-hydroxybenzoic acid is established as an index component, breaking the defect that Shujincao and its related preparations have no index component to reflect the overall content. The internal quality of Shujincao and its related preparations can be controlled from the whole and macroscopically, and the efficacy of the drug is ensured, so that the medicinal materials and their related preparations have obtained more regular quality control. The present application method has good stability, high precision, good reproducibility, convenience and easy to master.

[0185] The above is only the preferred embodiment of the present application, and it should be noted that for ordinary skilled persons in the technical field, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for constructing a high-performance liquid chromatography content determination of Sushencao, comprising the following steps: respectively, and the content is determined by high-performance liquid chromatography, and the determination is completed; the test sample solution is prepared according to the following method: The test sample and 70% ethanol are mixed, the weight is determined, and the mixture is refluxed or ultrasonically treated, cooled, and weighed again, and the lost weight is made up with 70% ethanol, shaken, filtered, and the filtrate is obtained; The control solution is 4-hydroxybenzoic acid solution; The chromatographic conditions of the high-performance liquid chromatography are as follows: The chromatographic column is a C18 column; The mobile phase A is methanol; The mobile phase B is 0.1% acetic acid solution or 0.2% formic acid solution; The detection wavelength is 260 nm; Gradient elution; The gradient elution is specifically as follows: 0-40 min, the mobile phase A is 7-15%, and the mobile phase B is 93-85%.

2. The construction method of claim 1, wherein, The column temperature of the chromatographic column is 25-35 DEG C.

3. The construction method of claim 1, wherein, The flow rate of the mobile phase is 0.8-1.2 ml / min, the injection amount is 8-10 mu l, and the theoretical plate number calculated according to the peak of 4-hydroxybenzoic acid should not be less than 5000.

4. The construction method of claim 1, wherein, The control solution is prepared according to the following method: The 4-hydroxybenzoic acid control sample is dissolved in 70% ethanol to obtain 4-hydroxybenzoic acid solution.

5. The construction method of claim 1, wherein, If the test sample is Sushencao standard decoction or Sushencao formula granules, the mass of the test sample and the volume of 70% ethanol are (0.05-1.00) g: 20 mL; If the test sample is Sushencao medicinal material or Sushencao decoction piece, the mass of the test sample and the volume of 70% ethanol are (0.2-2.0) g: 25 mL.

6. The construction method of claim 5, wherein, The ultrasonic treatment power is 580-620 W, the ultrasonic treatment frequency is 35-45 kHz, and the ultrasonic treatment time is 25-35 min.

7. The construction method of claim 1, wherein, The injection concentration of the 4-hydroxybenzoic acid solution is 1.0072-50.3600 mu g / ml.

8. A method for identifying Sarcandra glabra, characterized in that, The detection is performed by using the construction method of any one of claims 1-7, and the analysis and detection results are obtained.

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