Method for rapidly measuring content of lysimachia christinae hance granules

Abstract

The invention provides a method for rapidly determining the content of lysimachia christinae hance granules, and a detection sample preparation method of the lysimachia christinae hance granules comprises the following steps: taking the lysimachia christinae hance granules, adding water for dissolving, loading a sample of a dissolved solution to a D101 type macroporous adsorption resin column, eluting with water until an eluent is colorless, then eluting with a methanol solution, collecting the methanol eluent, adding hydrochloric acid for hydrolysis, and collecting the eluent; and adding a methanol solution, and uniformly mixing to obtain a sample solution to be detected. According to methodology verification, the content of quercetin and kaempferol in the lysimachia christinae hance granules can be rapidly and accurately detected, and the method has actual application and popularization value.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for rapidly determining the content of Lysimachiae Herba Granules. Background Art

[0002] Lysimachiae Herba Granules are light brown to brownish - brown granules made with "Lysimachiae Herba" as the active ingredient, supplemented with sucrose and dextrin; they taste sweet and slightly bitter, and their main functions are to clear away damp - heat, promote urination, and reduce swelling. Clinically, they are mainly used to treat heat strangury, sand strangury, painful and hesitant urination, jaundice with red urine, carbuncles and furuncles, snake bites, hepatolithiasis, urinary calculi and other diseases.

[0003] Currently, there are 4 approval numbers for Lysimachiae Herba Granules, and their quality control follows national drug standards, such as the "National Drug Standard" WS - 11446(ZD - 1446) - 2002 - 2013, and YBZ - PFKL - 2021072. These standards have detailed regulations on the content determination of Lysimachiae Herba formula granules to ensure the stability and consistency of product quality. Through experimental verification, when determining quercetin and kaempferol in Lysimachiae Herba Granules according to the YBZ - PFKL - 2021072 standard, since the main active ingredients quercetin and kaempferol in Lysimachiae Herba Granules often exist in the form of flavonoid glycosides (such as quercitrin), it is necessary to convert the conjugated flavonoid glycosides into free aglycones (such as quercetin and kaempferol) through acid hydrolysis method, so as to significantly increase the detectable amount of the target compounds quercetin and kaempferol. However, before acid hydrolysis, the excipients are not removed by column chromatography, which affects the subsequent hydrolysis to release quercetin and kaempferol, resulting in a low detection result. Moreover, the excipients are not removed by column chromatography in the sample pretreatment stage, which will affect the performance of the HPLC chromatographic column, especially it is easy to cause blockage or contamination after long - term use, shorten the service life of the chromatographic column, and increase the detection cost. Therefore, the WS - 11446(ZD - 1446) - 2002 - 2013 standard is widely used in actual quality control. However, when pretreating samples, it is difficult for the solution to pass through the polyamide column of the chromatographic column, resulting in a long elution time, and thus the preparation of the entire test solution takes a long time, and the repeatability of the chromatographic analysis results is not ideal. Summary of the Invention

[0004] To solve the above - mentioned problems, the present invention provides a method for preparing a detection sample of Lysimachiae Herba Granules, which includes the following steps:

[0005] Take Lysimachiae Herba Granules, dissolve them in water, load the dissolved solution onto a D101 macroporous adsorption resin column, elute with water until the eluate is colorless, then elute with a methanol solution, collect the methanol eluate, add hydrochloric acid for hydrolysis, and then mix with a methanol solution to obtain the sample solution to be tested.

[0006] Further, the mass-volume ratio of the Lysimachiae Herba granule, the water for dissolution, the methanol solution for elution, and hydrochloric acid is 2-4 g: 30 ml: 60 ml: 12 ml; each 100 ml of the test sample solution contains 12 ml of hydrochloric acid.

[0007] Furthermore, the concentration of the methanol solution is 70-90%, preferably 80%.

[0008] Further, the elution flow rate is 2-4 ml / min, preferably 3 ml / min.

[0009] Further, the hydrolysis temperature is 90 °C and the time is 1 hour.

[0010] Further, the inner diameter of the D101 macroporous adsorption resin column is 1.5-2.0 cm and the column height is 12 cm.

[0011] The present invention also provides a method for determining the content of Lysimachiae Herba granule, which comprises the following steps:

[0012] (1) Take quercetin reference substance and kaempferol reference substance, and prepare a reference substance solution with 70-90% methanol solution.

[0013] (2) Respectively take the reference substance solution obtained in step (1) and the aforementioned obtained test sample solution and inject them into a high performance liquid chromatograph, and the chromatographic conditions are as follows:

[0014] Chromatographic column: packed with octadecylsilane chemically bonded silica gel; mobile phase: methanol-0.4% phosphoric acid solution with a volume ratio of 50:50.

[0015] (3) Calculate the contents of quercetin reference substance and kaempferol in the Lysimachiae Herba granule by the external standard method based on the peak area.

[0016] Further, the concentration of the methanol solution in step (1) is 80%; each 1 ml of the reference substance solution contains 1-5 μg of quercetin and kaempferol respectively.

[0017] Further, the chromatographic column in step (2) is a waters sunfire C18 chromatographic column, 4.6×250 mm, 5 μm; the flow rate in the chromatographic conditions is 1 ml / min, the column temperature is 35 °C, the detection wavelength is 360 nm, and the injection volume is 10-20 μl.

[0018] The present invention deeply studies different chromatography columns and their specifications, and finds that when using a polyamide column to filter and purify the Lysimachia christinae Hance Granules solution, not only does the elution time become long, but it also causes the loss of quercetin and kaempferol, affecting the stability and accuracy of detection. Only a D101 macroporous adsorption resin column with specific models and specifications can shorten the pretreatment time of Lysimachia christinae Hance Granules and overcome the loss problem during the separation and purification of quercetin and kaempferol, improving the accuracy of detection. Through methodological verification, the method of the present invention can quickly and accurately detect the contents of quercetin and kaempferol in Lysimachia christinae Hance Granules, and has practical popularization and application value.

[0019] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can be made.

[0020] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0021] Figure 1 Chromatogram of the reference substance solution;

[0022] Figure 2 Chromatogram of the test solution (through the polyamide column);

[0023] Figure 3 Chromatogram of the test solution (through the macroporous resin column);

[0024] Figure 4 Chromatogram of the negative sample solution. Detailed Description of the Invention

[0025] Example 1 Determination of the Content of Lysimachia christinae Hance Granules

[0026] 1) Preparation of the test solution

[0027] Take 4 g of finely ground Lysimachia christinae Hance Granules, accurately weigh, add 30 ml of hot water, stir to dissolve, cool, and pass through a D101 macroporous adsorption resin column (inner diameter 1.5 - 2.0 cm, column height 12 cm) at a flow rate of 3 ml / min. Wash with water until the eluate is colorless, discard the aqueous solution, then elute with 60 ml of 80% methanol solution at a flow rate of 3 ml / min, collect the eluate, place it in a stoppered conical flask, add 12 ml of hydrochloric acid, heat and hydrolyze in a 90°C water bath for 1 hour, quickly cool, quantitatively transfer to a 100 ml volumetric flask, wash the container with 80% methanol solution, combine the washing solution into the same volumetric flask, dilute to the mark with 80% methanol, shake well, filter, and take the subsequent filtrate, that is, obtain;

[0028] 2) Preparation of reference substance solution: Weigh quercetin reference substance and kaempferol reference substance accurately, add 80% methanol to make a mixed solution containing 2 μg of each reference substance per 1 ml, and that is ready.

[0029] 3) Respectively pipette 20 μl of the test solution and the reference substance solution into a high performance liquid chromatograph, with the chromatographic conditions as follows:

[0030] Chromatographic column: waters sunfire C18 chromatographic column (4.6×250 mm, 5 μm);

[0031] Mobile phase: methanol - 0.4% phosphoric acid solution (50:50);

[0032] Detection wavelength is 360 nm; column temperature is 35 °C, and the flow rate is 1 ml / min.

[0033] 4) Calculate the contents of quercetin and kaempferol by external standard method according to the peak areas.

[0034] The beneficial effects of the present invention are further illustrated by the following test examples:

[0035] Test Example 1 Research and verification of the rapid determination method for the content of Lysimachiae Herba Granules

[0036] 1. Sample source

[0037] A total of 10 batches of Lysimachiae Herba Granule samples from 3 enterprises were collected. The 3 enterprises are Chengdu Dikang Pharmaceutical Co., Ltd., Chongqing Kerui Pharmaceutical (Group) Co., Ltd., and Chongqing Heping Pharmaceutical Co., Ltd. The negative sample was provided by Chengdu Dikang Pharmaceutical Co., Ltd.

[0038] 2. Instruments and reagents

[0039] Instruments: Thermo scientific Vanquish high performance liquid chromatograph (Thermo Fisher Scientific (China) Co., Ltd.), Waters-ARC-2998 high performance liquid chromatograph (Waters Technology (Shanghai) Co., Ltd.), Agilent 1260 high performance liquid chromatograph (Agilent Technologies (China) Co., Ltd.); XPE26 electronic balance (Mettler Toledo AG, Switzerland); AUX-220 electronic balance (Shimadzu Corporation, Japan), etc.

[0040] Reference substances: Quercetin reference substance (99.1%, 100081-201610), kaempferol reference substance (93.2%, 110861-202013), provided by the National Institutes for Food and Drug Control.

[0041] Lysimachiae Herba Granules (Chengdu Dikang Pharmaceutical Co., Ltd., batch number 230201).

[0042] Macroporous adsorption resin D101: Chengdu Kelong Chemical Co., Ltd.; Sinopharm Chemical Reagent Co., Ltd. (Shanghai Reagent); Tianjin Bailuns Biotechnology Co., Ltd.

[0043] Polyamide: Jiangsu Changfeng Chemical Co., Ltd.

[0044] The methanol in the mobile phase is of chromatographic purity, and the rest of the reagents and drugs are of analytical purity.

[0045] 3.

Assay

[0046] Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler (waters sunfire C18 chromatographic column, 4.6×250mm, 5μm); use methanol - 0.4% phosphoric acid solution (50:50) as the mobile phase; the detection wavelength is 360nm; the column temperature is 35°C, and the flow rate is 1ml / min. The number of theoretical plates calculated by quercetin should not be less than 2500.

[0047] Preparation of reference substance solution Weigh an appropriate amount of quercetin reference substance and kaempferol reference substance accurately, add 80% methanol to make a mixed solution containing 2μg of each per 1ml, and you will get it.

[0048] Preparation of test solution Take the Lysimachiae Herba granules under the difference in filling quantity, grind them finely, weigh accurately, add hot water, stir to dissolve, cool, pass through a chromatographic column (flow rate 3ml / min), wash with water until the eluate is colorless, discard the aqueous solution, then elute with 80% methanol solution (flow rate 3ml / min), collect the eluate, place it in a stoppered conical flask, add hydrochloric acid, heat and hydrolyze in a 90°C water bath for 1 hour, quickly cool, quantitatively transfer it to a volumetric flask, wash the container with 80% methanol solution, combine the washing solution into the same volumetric flask, dilute to the mark with 80% methanol, shake well, filter, and take the subsequent filtrate, and you will get it.

[0049] Determination method Accurately pipette 10 - 20μl of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and determine to get it.

[0050] Each bag of Lysimachiae Herba granules contains Lysimachiae Herba with the total amount of quercetin (C 15 H 10 O7) and kaempferol (C 15 H 10 O6) not less than 0.80mg.

[0051] 4. Preparation of test solution

[0052] 4.1 Investigation of the type of chromatographic column

[0053] Take an appropriate amount of Lysimachiae Herba Granules (batch number 230201), grind it finely, take 9 g, weigh it accurately, add 30 ml of hot water, stir to dissolve, cool, and pass through the following chromatographic columns at a flow rate of 3 ml / min (polyamide, 80 - 100 mesh, 5 g; D101 macroporous adsorption resin column, height 12 cm, inner diameter 2.0 cm), wash with water until the eluate is colorless, discard the aqueous solution, then elute with 150 ml of 80% methanol solution (flow rate 3 ml / min), collect the eluate, place it in a stoppered conical flask, add 30 ml of hydrochloric acid, heat and hydrolyze in a 90°C water bath for 1 hour, quickly cool, quantitatively transfer to a 250 ml volumetric flask, wash the container with 80% methanol solution, combine the washings into the same volumetric flask, dilute to the mark with 80% methanol, shake well, filter, and take the continuous filtrate to obtain the sample solution. Determine according to the selected chromatographic conditions, and the results are shown in Table 1.

[0054] Table 1 Investigation Results of Chromatographic Column Types

[0055]

[0056] Test results: For the test solution prepared by the D101 macroporous adsorption resin column, the contents of quercetin and kaempferol determined are higher than those of the test solution prepared by the polyamide column, and the time consumed is only one-sixth of that of the test solution prepared by the polyamide column. Selecting the D101 macroporous adsorption resin for the preparation of the test solution can shorten the detection time and improve the detection efficiency.

[0057] 4.2 Investigation of the Inner Diameter of the Chromatographic Column

[0058] Take an appropriate amount of Lysimachiae Herba Granules (batch number 230201), grind it finely, take 9 g, weigh it accurately, add 30 ml of hot water, stir to dissolve, cool, and pass through the D101 macroporous adsorption resin column (inner diameters are 1.0 cm, 1.5 cm, and 2.0 cm respectively; column height 12 cm, flow rate 3 ml / min), wash with water until the eluate is colorless, discard the aqueous solution, then elute with 150 ml of 80% methanol solution (flow rate 3 ml / min), collect the eluate, place it in a stoppered conical flask, add 30 ml of hydrochloric acid, heat and hydrolyze in a 90°C water bath for 1 hour, quickly cool, quantitatively transfer to a 250 ml volumetric flask, wash the container with 80% methanol solution, combine the washings into the same volumetric flask, dilute to the mark with 80% methanol, shake well, filter, and take the continuous filtrate to obtain the sample solution. Determine according to the selected chromatographic conditions. Compare the effects of different inner diameters of the resin column on the preparation of the test solution and the results of content determination respectively, and the results are shown in Table 2.

[0059] Table 2 Effects of the Inner Diameter of the Chromatographic Column on the Preparation of the Test Solution and the Results of Content Determination

[0060]

[0061] Test results: When the inner diameter of the column was 1.0 cm, the contents of quercetin and kaempferol in the sample were lower than those in the other groups, and the time consumption was longer. For the test solution prepared with a column inner diameter of 1.5 cm and 2.0 cm, the contents of quercetin and kaempferol were the highest and the time consumption was shorter. Therefore, a column inner diameter of 1.5 - 2.0 cm was selected.

[0062] 4.3 Investigation of column height

[0063] Take an appropriate amount of Lysimachiae Herba granule sample (batch number 230201), grind it finely, take 9 g, weigh it accurately, add 30 ml of hot water, stir to dissolve, cool, pass through a D101 macroporous adsorption resin column (flow rate 3 ml / min), wash with water until the eluate is colorless, discard the aqueous solution, then elute with 150 ml of 80% methanol solution (flow rate 3 ml / min), collect the eluate, place it in a stoppered conical flask, add 30 ml of hydrochloric acid, heat and hydrolyze in a 90 °C water bath for 1 hour, cool quickly, quantitatively transfer it to a 250 ml volumetric flask, wash the container with 80% methanol solution, combine the washing solution into the same volumetric flask, dilute to the mark with 80% methanol, shake well, filter, and take the continuous filtrate to obtain the test solution. Carry out the determination according to the selected chromatographic conditions. Compare the effects of different column heights on the preparation of the test solution and the results of content determination. The results are shown in Table 3.

[0064] Table 3 Effects of column height on the preparation of the test solution and the results of content determination

[0065]

[0066] Test results: The contents of quercetin and kaempferol measured in the 4 groups of test solutions were basically the same. When the column height was 15 cm and 20 cm, the preparation time of the test solution was longer. Therefore, a column inner diameter of 1.5 - 2.0 cm and a column height of 12 cm were selected.

[0067] 4.4 Investigation of macroporous adsorption resin types

[0068] Take an appropriate amount of Lysimachiae Herba granule sample (batch number 230201), grind it finely, take 9 g, weigh it accurately, add 30 ml of hot water, stir to dissolve, cool, and pass through different types of macroporous adsorption resin columns in Table 5 (column height 12 cm; inner diameter 2.0 cm) at a flow rate of 3 ml / min respectively, wash with water until the eluate is colorless, discard the aqueous solution, then elute with 150 ml of 80% methanol solution (flow rate 3 ml / min), collect the eluate, place it in a stoppered conical flask, add 30 ml of hydrochloric acid, heat and hydrolyze in a 90 °C water bath for 1 hour, cool quickly, quantitatively transfer it to a 250 ml volumetric flask, wash the container with 80% methanol solution, combine the washing solution into the same volumetric flask, dilute to the mark with 80% methanol, shake well, filter, and take the continuous filtrate to obtain the test solution. Carry out the determination according to the selected chromatographic conditions. The results are shown in Table 4.

[0069] Table 4 Investigation Results of Macroporous Adsorption Resin Models

[0070]

[0071] Test Results: For the macroporous adsorption resin columns of the above four models, there is no difference in the time required for the prepared test solution to pass through the column. Among them, for the test solution prepared with the D101 macroporous adsorption resin column, the measured contents of quercetin and kaempferol are significantly higher than those of the test solutions prepared with macroporous adsorption resin columns of other models. According to the Chinese Pharmacopoeia and relevant guiding principles, for drug components with a content less than 0.5%, the RSD should be less than 10%. If the RSD value is greater than 10%, the test results are considered not to be systematic errors. For the test solutions obtained by treating samples with the above four different models of macroporous adsorption resin columns, the RSD of the quercetin content by high-performance liquid chromatography quantitative analysis is 19.00%, and the RSD of the kaempferol content is 17.00%, both exceeding the error range, indicating that different models of macroporous adsorption resin for treating Lysimachiae Herba samples will affect the accuracy of detection. When selecting the D101 macroporous adsorption resin for the preparation of the test solution, it can effectively extract the substances to be measured in the test sample, avoid the loss of the substances to be measured, and ensure the accuracy of detection.

[0072] 4.5 Investigation of D101 Macroporous Adsorption Resins of Different Brands

[0073] Take 3 groups of the same batch of samples (230201), each group takes 9 g of samples, add 30 ml of hot water respectively, stir to dissolve, cool, and then add them to three brands of D101 macroporous adsorption resin columns (inner diameter 2.0 cm, column height 12 cm, control the flow rate at 3 ml / min) respectively. Wash with water until the eluate is colorless, discard the aqueous solution, then elute with 150 ml of 80% methanol solution (flow rate 3 ml / min), collect the eluate, place it in a stoppered conical flask, add 30 ml of hydrochloric acid, heat and hydrolyze in a 90°C water bath for 1 hour, quickly cool, quantitatively transfer to a 250 ml volumetric flask, wash the container with 80% methanol solution, combine the washing solutions into the same volumetric flask, dilute to the mark with 80% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution. Determine according to the selected chromatographic conditions, and the results are shown in Table 5.

[0074] Table 5 Influence of D101 Macroporous Adsorption Resins of Different Brands on the Preparation of Test Solutions and Content Determination Results

[0075]

[0076] Manufacturers of D101 Macroporous Adsorption Resin: 1. Chengdu Kelong Chemical Co., Ltd., batch number 2022103101; 2. Sinopharm Chemical Reagent Co., Ltd. (Shanghai Test), batch number 20221206; 3. Tianjin Bailuns Biotechnology Co., Ltd., 20230213

[0077] Test results: When using macroporous adsorption resins of different brands of D101 type, the preparation time of the test solution was basically the same. The contents of quercetin and kaempferol measured in the prepared test solutions were basically the same, and the RSDs were all less than 10%, ensuring the accuracy of the detection. Therefore, the brand of D101 type macroporous adsorption resin had basically no influence on the preparation of the test solution for content determination.

[0078] 4.6 Investigation of the sampling amount of the test sample

[0079] Take an appropriate amount of Lysimachiae Herba Granules (batch number 230201), grind it finely, weigh 9 g, 4 g, and 2 g respectively with precision, add 30 ml of hot water, stir to dissolve, cool, and pass through a D101 type macroporous adsorption resin column (inner diameter 2.0 cm, column height 12 cm, flow rate 3 ml / min). The rest is the same as "3 Content determination - Preparation of the test solution" starting from "elute with water until the eluate is colorless, discard the aqueous solution". Specifically, when the sample is 9 g, elute with 150 ml of 80% methanol solution, 30 ml of hydrochloric acid, and a 250 ml volumetric flask; when the sample is 4 g and 2 g, elute with 60 ml of 80% methanol solution, 12 ml of hydrochloric acid, and a 100 ml volumetric flask. The prepared test solutions were determined according to the selected chromatographic conditions. The effects of different sampling amounts on the preparation of the test solution and the content determination results were compared respectively, and the results are shown in Table 6.

[0080] Table 6 Effects of sampling amount on the preparation of the test solution and the content determination results

[0081]

[0082] Test results: The contents of quercetin and kaempferol measured in the 4 groups of test solutions were not significantly different. Considering that the preparation of the test solutions with 4 g and 2 g of the sampling amount took less time, and also considering the subsequent steps of eluting with 80% methanol solution, hydrolysis, and volume fixation, a sampling amount of 4 g was selected.

[0083] To sum up, the preparation method of the test solution is as follows: Take the Lysimachiae Herba Granules under the difference in filling quantity, grind it finely, take 4 g, weigh it with precision, add 30 ml of hot water, stir to dissolve, cool, and pass through a D101 type macroporous adsorption resin column (inner diameter 1.5 - 2.0 cm, column height 12 cm, flow rate 3 ml / min). Elute with water until the eluate is colorless, discard the aqueous solution, then elute with 60 ml of 80% methanol solution (flow rate 3 ml / min), collect the eluate, place it in a stoppered conical flask, add 12 ml of hydrochloric acid, heat and hydrolyze in a 90 °C water bath for 1 hour, quickly cool, quantitatively transfer it to a 100 ml volumetric flask, wash the container with 80% methanol solution, merge the washing liquid into the same volumetric flask, dilute it to the mark with 80% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0084] 5 Methodology investigation

[0085] 5.1 Investigation of linear range

[0086] Accurately weigh 10.288 mg of quercetin reference substance (content 99.1%) and place it in a 25-ml volumetric flask. Add 80% methanol to dissolve and dilute to the mark, shake well to obtain reference solution ①; accurately weigh 11.055 mg of kaempferol reference substance (content 93.2%) and place it in a 50-ml volumetric flask. Add 80% methanol to dissolve and dilute to the mark, shake well to obtain reference solution ②. Accurately measure 5 ml of reference solution ① and 2 ml of reference solution ②, place them in the same 10-ml volumetric flask, add 80% methanol to dilute to the mark, shake well to obtain the reference stock solution. Gradually dilute the reference stock solution into a series of concentration mixed reference solutions. Accurately measure 10 μl of each reference solution and inject it into the liquid chromatograph, record the peak area. Taking the reference substance concentration as the abscissa and the peak area as the ordinate, plot the standard curve. The regression equation of quercetin is: y = 0.6468x - 0.3407, and the correlation coefficient R 2 is 1.0000; the regression equation of kaempferol is: y = 0.6803x - 0.0530, and the correlation coefficient R 2 is 1.0000. The test results for standard curve plotting are shown in Table 7 and Table 8.

[0087] Table 7 Determination results of quercetin standard curve

[0088]

[0089] Table 8 Determination results of kaempferol standard curve

[0090]

[0091] Test results: Quercetin shows a good linear relationship in the concentration range of 0.3983 - 95.0365 μg / ml, and kaempferol shows a good linear relationship in the concentration range of 0.0805 - 41.2130 μg / ml.

[0092] 5.2 Precision test

[0093] Accurately measure 5 ml of the above reference solution ① and 2 ml of reference solution ②, place them in the same 50-ml volumetric flask, add 80% methanol to dilute to the mark, shake well to obtain the mixed reference solution. Take 10 μl and inject it continuously for 6 times to measure the peak area. The results show that the precision RSD of quercetin is 1.29%, and the precision RSD of kaempferol is 1.42%. It indicates that the instrument has good precision.

[0094] 5.3 Repeatability test

[0095] Appropriately weigh a proper amount of Lysimachiae Herba Granules sample (batch number 230201), grind it finely, accurately weigh 6 portions, and determine according to the established chromatographic conditions. The RSD of the repeatability of quercetin was 1.29%, and the RSD of the repeatability of kaempferol was 1.29%. It shows that the repeatability of this determination method is good.

[0096] 5.4 Stability test

[0097] Take the same test solution (batch number 230201), and inject samples for determination at 0, 2, 4, 8, 16, and 24 h at room temperature according to the established chromatographic conditions. The results show that the test solution is basically stable within 24 hours after preparation, meeting the requirements for content determination.

[0098] 5.5 Specificity test

[0099] According to the preparation process of Lysimachiae Herba Granules, prepare a negative control sample without Lysimachiae Herba, and prepare a negative sample solution according to the method under "3 Content determination - Preparation of test solution". Inject samples under the conditions of "3 Content determination - Chromatographic conditions and system suitability test". The results show that no chromatographic peaks were detected at the corresponding chromatographic peak positions of quercetin reference substance and kaempferol reference substance in the negative control sample, without interference. See Figure 4 .

[0100] 5.6 Accuracy test

[0101] Accurately weigh an appropriate amount of the sample with known content (batch number 230201), grind it finely, take 9 portions (the sampling amount is 50% of the sampling amount of the test sample), accurately add quercetin reference substance solution and kaempferol reference substance solution at 0.5, 1.0, and 1.5 times the content of each sample, and determine according to the content determination method. Calculate the recovery rate. The recovery rate of quercetin is between 86.37% and 94.74%, and the recovery rate of kaempferol is between 87.26% and 94.36%, basically meeting the requirements.

[0102] 6. Results

[0103] Determine 10 batches of samples from 3 production enterprises according to the established content determination method. The results are shown in Table 9. The chromatograms of the reference substance and the test sample are shown in Figure 1 and Figure 3 ; the chromatogram with a polyamide column as the chromatographic column as a control is shown in Figure 2 .

[0104] Table 9 Results of content determination of Lysimachiae Herba Granules

[0105]

[0106] Based on the content determination results of multiple batches of Lysimachiae Herba Granules, they all meet the requirements of the established standard limits.

[0107] In summary, the present invention purifies the test sample of Lysimachiae Herba granules through a macroporous adsorption resin column of D101 type with specific models and specifications, which can shorten the pretreatment time of Lysimachiae Herba granules and overcome the loss problems of quercetin and kaempferol during the separation and purification process, thereby improving the accuracy of detection. Through methodological verification, the method of the present invention can quickly and accurately detect the contents of quercetin and kaempferol in Lysimachiae Herba granules, and has practical popularization and application value.

Claims

1. A method for configuring a sample for testing Lysimachia chinensis granules, characterized in that: It includes the following steps: Take the Lysimachia chinensis granules, add water to dissolve them, load the dissolved solution onto a D101 macroporous adsorption resin column, elute with water until the eluent is colorless, then elute with a methanol solution, collect the methanol eluent, add hydrochloric acid to hydrolyze it, and then add the methanol solution to mix well to obtain the sample solution to be tested.

2. The detection sample configuration method according to claim 1, characterized in that: The mass volume ratio of the Lysimachia sibiricum particles to the dissolving water, the eluting methanol solution and the hydrochloric acid is 2-4 g: 30 ml: 60 ml: 12 ml; and each 100 ml of the sample solution to be tested contains 12 ml of hydrochloric acid.

3. The detection sample configuration method according to claim 1 or 2, characterized in that: The concentration of the methanol solution is 70-90%, preferably 80%.

4. The detection sample configuration method according to claim 1, characterized in that: The elution flow rate is 2-4 ml / min, preferably 3 ml / min.

5. The detection sample configuration method according to claim 1, characterized in that: The hydrolysis temperature is 90° C. and the time is 1 hour.

6. The detection sample configuration method according to claim 1, characterized in that: The inner diameter of the D101 macroporous adsorption resin column is 1.5-2.0 cm, and the column height is 12 cm.

7. A method for determining the content of Lysimachia chinensis granules, characterized in that: It includes the following steps: (1) Take quercetin reference substance and kaempferol reference substance, add 70-90% methanol solution to prepare reference substance solution; (2) The reference solution obtained in step (1) and the test sample solution obtained in claim 1 are respectively injected into a high performance liquid chromatograph under the following chromatographic conditions: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: methanol-0.4% phosphoric acid solution in a volume ratio of 50:50; (3) The contents of quercetin and kaempferol in Houttuynia cordata granules were calculated by the external standard method based on the peak area.

8. The determination method according to claim 7, characterized in that: The concentration of the methanol solution in step (1) is 80%; and each 1 ml of the reference solution contains 1 to 5 μg of quercetin and kaempferol.

9. The assay method according to claim 7, characterized in that: The chromatographic column in step (2) is a watersunfire C18 chromatographic column, 4.6×250 mm, 5 μm.

10. The determination method according to claim 7, characterized in that: The chromatographic conditions in step (2) are as follows: the flow rate is 1 ml / min, the column temperature is 35° C., the detection wavelength is 360 nm, and the injection volume is 10 to 20 μl.

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