Method for determining contents of multiple components in modified Qing'e pills

Abstract

The invention belongs to the technical field of pharmaceutical analysis and quality control, and particularly relates to a method for determining the content of multiple components in modified Qing'e pills. According to the method for determining the content of the multiple components in the modified Qing'e pill, the high performance liquid chromatography technology is utilized to establish the method for determining the content of the multiple components in the modified Qing'e pill, and the content of the multiple components in the modified Qing'e pill is determined by optimizing the characteristics of the chromatography technology, especially optimizing a mobile phase elution program. The method can be used for simultaneously determining various chemical components in the modified Qing'e pill, and has the advantages of simplicity and convenience in operation, reliable detection result, high stability and precision, good repeatability, energy conservation and environmental protection.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and quality control, and specifically relates to a method for determining the contents of multiple components in a modified Qing'e pill. Background Art

[0002] Qing'e Pills have the effect of tonifying the kidney and strengthening the waist. It is a commonly used medicine for the treatment of kidney deficiency and low back pain. It is clinically used for the treatment of kidney deficiency and low back pain, sitting difficulties, weak knees and other symptoms. The prescription composition of Qing'e Pills included in the "Chinese Pharmacopoeia" (2020 edition) includes: 480g of Eucommia (fried with salt), 240g of Psoralea corylifolia (fried with salt), 150g of walnut kernels (fried), and 120g of garlic. Beijing Tongrentang uses Qing'e Pills as the basic prescription, adjusts the original prescription and develops an exclusive product, Jiawei Qing'e Pills, whose prescription composition includes Psoralea corylifolia (fried with salt), walnut kernels, Morinda officinalis (processed), Cistanche deserticola (fried with wine), Eucommia ulmoides (charcoal), frankincense (fried with vinegar), and myrrh (fried with vinegar). Jiawei Qing'e Pills have the effects of tonifying the kidney, dispelling cold, and relieving pain. They are clinically used to treat low back and leg pain, frequent urination, and lower abdominal cold pain caused by kidney meridian deficiency and cold. The pill-making process of Jiawei Qing'e Pills is as follows: Psoralea corylifolia (fried with salt), walnut kernel, Morinda officinalis (processed), Cistanche deserticola (roasted with wine), Eucommia ulmoides (charcoal), frankincense (roasted with vinegar), and Myrrha (roasted with vinegar). After the above drugs are crushed, sieved and mixed, refined honey is directly added to make pills.

[0003] At present, the implementation standard of Jiawei Qing'e Pills is the Chinese Medicine Formula Preparation (Volume 11) WS3-B-2123-96 of the Drug Standards of the Ministry of Health of the People's Republic of China, and it has not yet been included in the Chinese Pharmacopoeia. At present, the research on the quality evaluation of Jiawei Qing'e Pills is mainly the establishment method of the fingerprint spectrum of the compound medicine recorded in the Chinese patent CN117330662A, which discloses the fingerprint spectrum construction method suitable for Jiawei Qing'e Pills. In addition, Ai Lu et al. also recorded the method for determining the content of psoralen and isopsoralen in Jiawei Qing'e Pills in the article "Determination of the content of psoralen and isopsoralen in Jiawei Qing'e Pills by high performance liquid chromatography", but the study did not identify most of the medicinal materials, and there are certain limitations.

[0004] Therefore, this field is looking forward to developing a method that can detect the content of multiple components in Jiawei Qing'e Pills, and then establishing a complete quality evaluation system, which will have positive significance for ensuring drug safety and promoting the process of drugs being included in the "Chinese Pharmacopoeia", thereby ensuring the safety and effectiveness of drugs. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a method for determining the content of multiple components in the modified Qing'e Pills. The method establishes a method for determining the content of 9 components in the modified Qing'e Pills, which provides a solid technical support and scientific basis for the quality evaluation of the modified Qing'e Pills.

[0006] The second technical problem to be solved by the present invention is to provide an application of the method for determining the contents of multiple components in the above-mentioned Jiawei Qing'e Pills in the field of quality control of Jiawei Qing'e Pills.

[0007] In order to solve the above technical problems, the present invention discloses a method for determining the contents of multiple components in a flavored Qing'e pill, comprising the steps of performing high performance liquid chromatography on a test solution of the flavored Qing'e pill;

[0008] The chromatographic conditions include: using octadecylsilane bonded silica gel as a filler, using acetonitrile as a mobile phase A, using 0.1% formic acid aqueous solution (v / v) as a mobile phase B, and performing gradient elution according to the following procedure:

[0009] 0-8min, A:B is 10%→13%:90%→87%;

[0010] 8-15min, A:B 13%→36%:87%→64%;

[0011] 15-20min, A:B 36%→40%:64%→60%;

[0012] 20-45min, A:B is 40%→75%:60%→25%;

[0013] 45-47min, A:B 75%→10%:25%→90%;

[0014] 47-60min, A:B is 10%:90%.

[0015] Specifically, in the method for determining the contents of multiple components in the modified Qing'e Pills, in the high performance liquid chromatography detection step, the chromatographic conditions further include:

[0016] Column temperature 30-40°C, preferably 35°C; and / or,

[0017] Flow rate 0.1-0.5 ml / min, preferably 0.3 ml / min; and / or,

[0018] Detection wavelengths are 250nm and 350nm.

[0019] Specifically, the method for determining the content of multiple components in the flavored Qing'e Pills, the method for preparing the test solution comprises: taking the flavored Qing'e Pills to be tested sample, adding an alcohol solution to extract, and collecting the extract.

[0020] Specifically, the method for determining the content of multiple components in the modified Qing'e pills and the method for preparing the test solution are as follows:

[0021] The alcohol solution comprises a methanol solution, preferably a 50%-100% methanol solution (v / v); and / or,

[0022] The extraction step comprises ultrasonic extraction; preferably, the ultrasonic extraction power is 300-600W, and the ultrasonic extraction frequency is 20-60kHz; and / or,

[0023] The extraction time of the extraction step is 15-45 min; and / or,

[0024] The mass ratio of the sample to be tested of the flavored Qing'e Pills to the volume ratio of the alcohol solvent is (0.5-1.5):(25-50), wherein the mass unit of the sample to be tested of the flavored Qing'e Pills is g, and the volume unit of the alcohol solvent is ml.

[0025] Specifically, the method for determining the content of multiple components in the flavored Qing'e pills, the method for preparing the test solution further comprises the step of adding the flavored Qing'e pills to be tested sample to diatomaceous earth for mixing;

[0026] Preferably, the mass ratio of the sample to be tested to the diatomaceous earth is 1-3:1.

[0027] Specifically, the method for determining the content of multiple components in the modified Qing'e pills, the detection indicators of the modified Qing'e pills include: at least one of psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavones, psoralenine, psoralenidine, psoralen dihydroflavone methyl ether or isopsoralen chromene chalcone.

[0028] Specifically, the method for determining the content of multiple components in the modified Qing'e pills also includes the step of fitting a linear equation for the content detection of the detection index.

[0029] Specifically, the method for determining the content of multiple components in the modified Qing'e Pills, in which the linear equations of different detection indicators in the modified Qing'e Pills include:

[0030] Psoralen: y=20245x-4418.1; and / or,

[0031] Isopsoralen: y=20661x-187.23; and / or,

[0032] Psoralen: y=45454x-18464; and / or,

[0033] Isopsoralen: y=48777x-15625; and / or,

[0034] New psoralea corylifolia isoflavones: y=34253x-3344.6; and / or,

[0035] Psoralen: y=60038x-2860; and / or,

[0036] Psoralen: y=22522x-454.04; and / or,

[0037] Psoralea corylifolia methyl ether: y=7643.8x-2270.4; and / or,

[0038] Isopsoralea corylifolia chromone: y = 35692x-508.59;

[0039] Where x is the injection concentration and y is the peak area.

[0040] The invention also discloses the application of the method for determining the contents of multiple components in the modified Qing'e pills in the field of quality detection and control of the modified Qing'e pills.

[0041] The present invention also discloses a quality control method for the modified Qing'e pills, comprising the steps of determining the contents of multiple components in the modified Qing'e pills according to the method;

[0042] Preferably, the detection components of the modified Qing'e Pills include at least one of psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavones, psoralenine, psoralenidine, psoralen dihydroflavone methyl ether or isopsoralen chromene chalcone.

[0043] The method for determining the contents of multiple components in the modified Qing'e pills of the present invention is established by utilizing high performance liquid chromatography technology. By optimizing the characteristics of the chromatographic technology, especially the optimization of the mobile phase elution program, the method can simultaneously determine the multiple chemical components in the modified Qing'e pills. The method has the advantages of simple operation, reliable detection results, high stability and precision, good repeatability, energy saving and environmental protection.

[0044] The method for determining the contents of multiple components in the modified Qing'e pills of the present invention can comprehensively and effectively control the quality of the modified Qing'e pills, thereby ensuring the stability, reliability and safety of the product, and providing a solid technical support and scientific basis for the quality evaluation of the modified Qing'e pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0046] Figure 1 The full wavelength (200-400nm) scanning results of each reference substance in Example 2; wherein the abscissa is nanometers and the ordinate is AU;

[0047] Figure 2 This is the chromatogram of the negative control sample in Example 3; wherein the abscissa is minutes and the ordinate is AU;

[0048] Figure 3 The chromatograms (250 nm) of the samples under different elution gradients in Example 4 are shown in FIG. 1 , wherein the horizontal axis is minutes and the vertical axis is AU.

[0049] Figure 4 The chromatograms of the reference substance and the test substance in Example 9; wherein the abscissa is time (min) and the ordinate is signal (mV). DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following embodiments. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention does not particularly limit the contents.

[0051] In the following embodiments of the present invention, the instrument and reagent information include:

[0052] Instruments: Waters ACQUITY ultra-high performance liquid chromatograph (Waters Technology Co., Ltd., USA); Mettler Toledo XP205 electronic balance (Mettler Toledo, Switzerland); Mettler Toledo ML204 electronic balance (Mettler Toledo, Switzerland); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); high-speed universal pulverizer (Tianjin Test Instrument Co., Ltd.);

[0053] Chromatographic column: Agilent EclipsePlus C18 (2.1×100 mm, 1.8 μm) (Agilent Technology Co., Ltd., USA);

[0054] Reagents: acetonitrile and formic acid were of chromatographic grade, water was ultrapure water, methanol and ethanol were of analytical grade;

[0055] Test drugs: Jiawei Qing'e Pills (batch numbers: 18015062, 17015466, 17015978, 18015267, 21010064, 20010731, 22010758, 23010994, 22010759, 23011295, 23010993, 23011294, 23011293, 23010995) were provided by Beijing Tong Ren Tang Pharmaceutical Factory;

[0056] Reference substances: psoralen (batch number: 110739-201918, content: 99.6%, for content determination, China Food and Drug Inspection Institute); isopsoralen (batch number: 110738-202016, content: 99.4%, for content determination, China Food and Drug Inspection Institute); psoralen (batch number: MUST-23112401, content: 99.17%, for content determination); isopsoralen (batch number: MUST-23112402, content: 98.95%, for content determination); neopsoralen isoflavones (batch number: 520052- 201401, content: 99.6%, for content determination, China Food and Drug Inspection Institute); Psoralen (Batch No.: MUST-23112403, content: 99.32%, for content determination); Psoralen (Batch No.: MUST-23090207, content: 99.48%, for content determination); Psoralen dihydroflavonoid methyl ether (Batch No.: MUST-22112605, content: 98.95%, for content determination); Isopsoralen chromone chalcone (Batch No.: MUST-23060707, content: 99.17%, for content determination).

[0057] Example 1

[0058] The mother solution is configured as follows:

[0059] Take an appropriate amount of psoralen, place it in a 25ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.77mg / ml;

[0060] Take an appropriate amount of isopsoralen, place it in a 25 ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.78 mg / ml;

[0061] Take an appropriate amount of psoralen, place it in a 25ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.49mg / ml;

[0062] Take an appropriate amount of isopsoralen, place it in a 25 ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.51 mg / ml;

[0063] Take an appropriate amount of new psoralea corylifolia isoflavones, place it in a 25ml volumetric flask, add methanol to the mark, and obtain a solution with a concentration of 0.64mg / ml;

[0064] Take an appropriate amount of Psoralen, place it in a 25ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.79mg / ml;

[0065] Take an appropriate amount of Psoralea corylifolia, place it in a 100ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.21mg / ml;

[0066] Take an appropriate amount of psoralea corylifolia methyl ether, place it in a 25 ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.77 mg / ml;

[0067] Take an appropriate amount of Isopsoralea corylifolia chromone, place it in a 100ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.05mg / ml.

[0068] Example 2

[0069] This example selects the detection wavelength based on the mother solution prepared in Example 1.

[0070] The chromatographic conditions of this embodiment are as follows:

[0071] Chromatographic column: Agilent Eclipse Plus C18 (2.1×100 mm, 1.8 μm);

[0072] Acetonitrile was used as mobile phase A, 0.1% formic acid aqueous solution (v / v) was used as mobile phase B, and gradient elution was performed according to the elution program in Table 1 below;

[0073] Column temperature: 35 °C;

[0074] Flow rate: 0.3ml / min.

[0075] Table 1 Elution program

[0076] Time (min) Mobile phase A (%) Mobile phase B (%) 0～8 10→13 90→87 8～15 13→36 87→64 15～20 36→40 64→60 20～45 40→75 60→25 45～47 75→10 25→90 47～60 10 90

[0077] Take appropriate amount of the prepared mother solution and scan at full wavelength in the range of 200-400nm. The chromatograms of different types of mother solution are shown in the attached Figure 1 shown.

[0078] It can be seen that psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavone, psoralenin, psoralen, psoralen, psoralen dihydroflavonoid methyl ether can be detected simultaneously at 250nm, so the detection wavelength of 8 compounds such as psoralen is selected as 250nm. The maximum absorption wavelength of isopsoralen chromene chalcone is about 350nm, so the detection wavelength selected is 350nm.

[0079] In summary, the detection method of the present invention selects detection wavelengths of 250nm and 350nm.

[0080] Example 3

[0081] Preparation of reference solution

[0082] Based on the mother solution prepared in Example 1, a mixed reference solution was prepared by stepwise dilution, wherein the concentration of each reference solution was: psoralen 45.32 μg / ml; isopsoralen 46.70 μg / ml; psoralen 29.06 μg / ml; isopsoralen 30.46 μg / ml; new psoralen isoflavones 15.23 μg / ml; psoralen 6.25 μg / ml; psoralenidine 12.71 μg / ml; psoralen dihydroflavone methyl ether 19.06 μg / ml; and isopsoralen chromene chalcone 0.86 μg / ml.

[0083] Preparation of test solution

[0084] Take 3 pills of Jiawei Qing'e Pills (batch number 23010994), accurately weigh 26.3727g, add 13.1996g of diatomaceous earth (batch number 20090813), grind to obtain mixed powder. Take about 1.5g of the mixed powder, accurately weigh it, put it in a stoppered conical bottle, accurately add 25ml of methanol, weigh it, ultrasonically treat it for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and you have it.

[0085] Preparation of negative control samples

[0086] According to the proportion of Chinese medicinal ingredients in the prescription, negative control samples of the drug group not containing psoralea corylifolia, Morinda officinalis, Cistanche deserticola, Eucommia ulmoides, walnut kernel, frankincense, and myrrh were prepared respectively, and the test sample solution was prepared.

[0087] Under the chromatographic conditions described in Example 2, the chromatogram of the negative control sample is shown in the attached Figure 2 shown.

[0088] It can be seen that the detection results of the detection method of the present invention are free of interference.

[0089] Example 4

[0090] In this example, the ratio of the mobile phase was adjusted based on the chromatographic conditions described in Example 2.

[0091] Acetonitrile was used as mobile phase A, 0.1% formic acid-water (v / v) was used as mobile phase B, and elution was performed according to different gradients as shown in Table 2. The elution effect was evaluated using the separation effect as the evaluation index.

[0092] Table 2 Elution gradient program

[0093]

[0094] In this embodiment, the sample chromatograms (250nm) under different elution gradients are shown in the attached Figure 3 .

[0095] By comparing the separation effects of various chromatographic peaks under different elution gradients, it was found that the chromatographic peaks under the gradient 5 elution condition had high separation and good peak shape.

[0096] Example 5

[0097] This example is based on the chromatographic conditions described in Example 2, and on the basis of the sample preparation method of Example 3, to investigate the detection differences under different sample extraction solvents.

[0098] Extraction 1: Take about 1.5 g of the flavored Qing'e pills sample dispersed with diatomaceous earth, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, ultrasonicate for 30 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the product.

[0099] Extraction 2: Take about 1.5 g of the sample dispersed with diatomaceous earth, accurately weigh it, accurately add 25 ml of 70% methanol, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0100] Extraction three: Take about 1.5 g of the sample dispersed with diatomaceous earth, weigh it accurately, add 25 ml of 50% methanol accurately, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0101] The contents of the nine components in the three extraction solvents were determined according to the recorded chromatographic conditions. The results are shown in Table 3.

[0102] Table 3 Comparison of extraction solvents

[0103]

[0104] The experimental results showed that when the extraction solvent was methanol, the nine components were extracted more completely.

[0105] Example 6

[0106] This example is based on the chromatographic conditions described in Example 2, and on the basis of the sample preparation method of Example 3, to investigate the detection differences under different sample extraction methods.

[0107] Extraction 1: Take about 1.5 g of the sample dispersed with diatomaceous earth, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, ultrasonicate for 30 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0108] Extraction 2: Take about 1.5 g of the sample dispersed with diatomaceous earth, weigh it accurately, add 25 ml of methanol accurately, weigh it, heat and reflux for 30 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0109] The contents of the 9 components under the above two extraction methods were determined according to the recorded chromatographic conditions. The results are shown in Table 4.

[0110] Table 4 Comparison of extraction methods

[0111]

[0112] The experimental results show that both reflux extraction and ultrasonic extraction can completely extract all nine components in the sample. Ultrasonic extraction is more convenient, environmentally friendly, and can process multiple samples at the same time.

[0113] Example 7

[0114] This example is based on the chromatographic conditions described in Example 2, and on the basis of the sample preparation method of Example 3, to investigate the detection differences under different amounts of sample extraction solvent.

[0115] Extraction 1: Take about 1.5 g of the sample dispersed with diatomaceous earth, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, ultrasonicate for 30 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0116] Extraction 2: Take about 1.5 g of the sample dispersed with diatomaceous earth, weigh it accurately, put it in a stoppered conical flask, accurately add 50 ml of methanol, weigh it, ultrasonicate for 30 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0117] The contents of the nine components at the two extraction solvent dosages were determined according to the recorded chromatographic conditions. The results are shown in Table 5.

[0118] Table 5 Comparison of extraction solvent usage

[0119]

[0120] The experimental results show that when the amount of extraction solvent is 25ml, the nine components have been completely extracted.

[0121] Example 8

[0122] This example is based on the chromatographic conditions described in Example 2, and on the basis of the sample preparation method of Example 3, to investigate the detection differences under different sample extraction times.

[0123] Extraction 1: Take about 1.5 g of the sample dispersed with diatomaceous earth, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, ultrasonicate for 15 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0124] Extraction 2: Take about 1.5 g of the sample dispersed with diatomaceous earth, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, ultrasonicate for 30 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0125] Extraction three: Take about 1.5g of the sample dispersed with diatomaceous earth, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of methanol, weigh it, ultrasonicate for 45 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0126] The contents of the nine components at the three extraction times were determined according to the recorded chromatographic conditions. The results are shown in Table 6.

[0127] Table 6 Comparison of extraction time

[0128]

[0129] The experimental results showed that the above 9 components were completely extracted when the extraction time was 30 minutes.

[0130] Example 9

[0131] By optimizing the parameters in the above Examples 5-8, the extraction method determined in this Example is: take about 1.5 g of the sample dispersed with diatomaceous earth, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, ultrasonicate for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0132] The preparation method of the reference substance is the same as that of Example 3.

[0133] In this example, based on the chromatographic conditions described in Example 2, the test sample and reference solution were tested, and the chromatograms of the reference sample and the test sample at 250nm and 350nm were respectively Figure 4 As shown in (a)-(b); wherein, 1-psoralen; 2-isopsoralen; 3-psoralen; 4-isopsoralen; 5-neopsoralen isoflavone; 6-psoralenine; 7-psoralin; 8-psoralen dihydroflavone methyl ether); 9-isopsoralen chromene chalcone.

[0134] Example 10

[0135] This example investigates the linear relationship of the detection method for the modified Qing'e Pills.

[0136] Prepare the mother solutions of each reference substance according to the method in Example 1:

[0137] Take an appropriate amount of psoralen, place it in a 25ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.77mg / ml;

[0138] Take an appropriate amount of isopsoralen, place it in a 25 ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.78 mg / ml;

[0139] Take an appropriate amount of psoralen, place it in a 25ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.49mg / ml;

[0140] Take an appropriate amount of isopsoralen, place it in a 25 ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.51 mg / ml;

[0141] Take an appropriate amount of new psoralea corylifolia isoflavones, place it in a 25ml volumetric flask, add methanol to the mark, and obtain a solution with a concentration of 0.64mg / ml;

[0142] Take an appropriate amount of Psoralen, place it in a 25ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.79mg / ml;

[0143] Take an appropriate amount of Psoralea corylifolia, place it in a 100ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.21mg / ml;

[0144] Take an appropriate amount of psoralea corylifolia methyl ether, place it in a 25 ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.77 mg / ml;

[0145] Take an appropriate amount of Isopsoralea corylifolia chromone, place it in a 100ml volumetric flask, add methanol to the mark to obtain a solution with a concentration of 0.05mg / ml.

[0146] Take an appropriate amount of the above-mentioned reference substance mother solution, place it in a 10 ml volumetric flask, add methanol to make up to the scale, and prepare a mixed reference substance solution containing 113.37 μg / ml of psoralen, 116.81 μg / ml of isopsoralen, 72.69 μg / ml of psoralen, 76.21 μg / ml of isopsoralen, 38.09 μg / ml of psoralen isoflavones, 15.64 μg / ml of psoralen, 31.79 μg / ml of psoralen, 47.69 μg / ml of psoralen dihydroflavone methyl ether, and 2.16 μg / ml of isopsoralen chromone chalcone.

[0147] Accurately measure 3ml, 2ml and 1ml of the mixed reference solution respectively, place them in a 5ml volumetric flask, and add methanol to make up to the mark; accurately measure 0.5ml of the mixed reference solution respectively, place them in a 10ml and 20ml volumetric flask, and add methanol to make up to the mark; the mixed standard reference solution of a series of concentrations is obtained as follows:

[0148] psoralen 2.84, 5.72, 22.53, 45.32, 67.81, 113.37 μg / ml;

[0149] isopsoralen 2.93, 5.90, 23.22, 46.70, 69.88, 116.81 μg / ml;

[0150] psoralen 1.82, 3.67, 14.45, 29.06, 43.48, 72.69 μg / ml;

[0151] isopsoralen 1.91, 3.85, 15.15, 30.46, 45.59, 76.21 μg / ml;

[0152] New psoralea isoflavones 0.96, 1.92, 7.57, 15.23, 22.78, 38.09 μg / ml;

[0153] Psoralen 0.39, 0.79, 3.11, 6.25, 9.36, 15.64 μg / ml;

[0154] Psoralen 0.80, 1.60, 6.32, 12.71, 19.02, 31.79 μg / ml;

[0155] Psoralea corylifolia 1.20, 2.41, 9.48, 19.06, 28.53, 47.69 μg / ml;

[0156] Isopsoralen chromone chalcone 0.05, 0.11, 0.43, 0.86, 1.29, 2.16 μg / ml.

[0157] According to the detection method described in the above Example 2, 2 μl of the sample was injected for analysis and the peak area was determined. Linear regression was performed with the injection concentration (x) as the horizontal axis and the peak area (y) as the vertical axis. The regression equations of each reference substance were calculated and shown in Table 7 below.

[0158] Table 7 Linear relationship investigation

[0159] name Regression equation <![CDATA[R 2 ]]> Linear range (μg / ml) Psoralen y=20245x-4418.1 0.9995 2.84～113.37 Isopsoralen y=20661x-187.23 0.9996 2.93～116.81 Psoralen y=45454x-18464 0.9997 1.82～72.69 Isopsoralen y=48777x-15625 0.9997 1.91～76.21 New Psoralea corylifolia Isoflavones y=34253x-3344.6 0.9997 0.96～38.09 Psoralea corylifolia y=60038x-2860 0.9999 0.39～15.64 Psoralen y=22522x-454.04 0.9999 0.80～31.79 Psoralea corylifolia dihydroflavonoid methyl ether y=7643.8x-2270.4 0.9994 1.20～47.69 Isopsoralen chromene chalcone y=35692x-508.59 0.9999 0.05～2.16

[0160] It can be seen that the detection of each effective ingredient in the modified Qing'e Pills of the present invention has a linear fitting relationship.

[0161] Embodiment 11

[0162] This example performs a precision test based on the detection method described in the present invention.

[0163] Accurately pipette the test solution of flavored Qing'e Pills (batch number: 23010994), inject it into the liquid chromatograph, inject 6 times continuously, 2 μl each time, measure the peak area, and the results are shown in Table 8 below.

[0164] Table 8 Precision test results

[0165]

[0166] The results showed that the instrument precision was good under the chromatographic conditions.

[0167] Example 12

[0168] This example conducts a stability test based on the detection method described in the present invention.

[0169] Accurately pipette the test solution of flavored Qing'e Pills (batch number: 23010994) and measure according to the law at 0, 2, 4, 8, 12, 18 and 24 hours respectively. The results are shown in Table 9 below.

[0170] Table 9 Stability test results

[0171]

[0172]

[0173] The test results showed that the test solution remained stable within 24 hours after preparation.

[0174] Example 13

[0175] This example conducts a repeatability test based on the detection method described in the present invention.

[0176] Accurately weigh 6 portions of the same batch number (23010994) of flavored Qing'e pills to prepare the test solution. The test was carried out in parallel according to the method. The results are shown in Table 10.

[0177] Table 10 Repeatability test results

[0178]

[0179] The results showed that this method had good repeatability.

[0180] Embodiment 14

[0181] This example conducts an accuracy (sample recovery test) experiment based on the detection method of the present invention.

[0182] Accurately weigh 0.75g of the same batch number (23010994) of Qing'e pills with known content (containing psoralen 1.43mg / g, isopsoralen 1.01mg / g, psoralen 0.81mg / g, isopsoralen 0.78mg / g, new psoralen isoflavone 0.46mg / g, psoralen 0.16mg / g, psoralen 0.34mg / g, psoralen dihydroflavone methyl ether 0.59mg / g, isopsoralen chromone chalcone psoralen 0.02mg / g), and accurately add 28.56μg / ml of psoralen control solution , isopsoralen control solution 20.41μg / ml, psoralen control solution 16.72μg / ml, isopsoralen control solution 16.01μg / ml, new psoralen isoflavone control solution 8.99μg / ml, psoralen control solution 3.31μg / ml, psoralen control solution 7.83μg / ml, psoralen dihydroflavone methyl ether control solution 11.90μg / ml, isopsoralen chromone chalcone psoralen control solution 0.49μg / ml, and 25ml of a mixed control solution was prepared according to the test solution preparation method for determination.

[0183] The results are shown in Tables 11-19 below.

[0184] Table 11 Psoralen recovery results

[0185]

[0186] Table 12 Isopsoralen recovery results

[0187]

[0188] Table 13 Psoralen sample recovery results

[0189]

[0190]

[0191] Table 14 Isopsoralen sample recovery results

[0192]

[0193] Table 15 New Psoralea corylifolia isoflavones sample recovery results

[0194]

[0195] Table 16 Psoralea corylifolia sample recovery results

[0196]

[0197] Table 17 Psoralea corylifolia sample recovery results

[0198]

[0199] Table 18 Psoralea corylifolia dihydroflavonoid methyl ether sample recovery results

[0200]

[0201] Table 19 Isopsoralea corylifolia chromone sample recovery results

[0202]

[0203] The results show that this method has good accuracy.

[0204] Embodiment 15

[0205] In this example, 14 batches of modified Qing'e pills were assayed based on the detection method of the present invention.

[0206] Take about 26g of 14 batches of samples respectively, weigh accurately, add 13g of diatomaceous earth, weigh accurately, grind finely, and obtain mixed powder. Take about 1.5g of the sample dispersed with diatomaceous earth, weigh accurately, put it in a stoppered conical bottle, add 25ml of methanol accurately, weigh the weight, ultrasonicate for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain. Determine according to the above chromatographic conditions, and the results are shown in Table 20.

[0207] Table 20 Content determination results of 14 batches of modified Qing'e pills

[0208]

[0209]

[0210] The results showed that this method can be used to simultaneously determine the contents of multiple components in Jiawei Qing'e Pills.

[0211] In summary, the detection method for simultaneous quality control of multiple components in the modified Qing'e Pills described in the present invention can simultaneously carry out content determination of multiple chemical components in the modified Qing'e Pills, and the established content determination method has the advantages of being simple and reliable, having high stability and precision, good repeatability, and being energy-saving and environmentally friendly.

[0212] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for determining the contents of multiple components in modified Qing'e pills, characterized in that: The method comprises the steps of conducting high performance liquid chromatography on a test solution of the flavored Qing'e pills; The chromatographic conditions include: using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and 0.1 v / v% formic acid aqueous solution as a mobile phase B, and performing gradient elution according to the following procedure: 0-8min, A:B 10%→13%:90%→87%; 8-15min, A:B 13%→36%:87%→64%; 15-20min, A:B 36%→40%:64%→60%; 20-45min, A:B is 40%→75%:60%→25%; 45-47min, A:B 75%→10%:25%→90%; 47-60min, A:B is 10%:90%.

2. The method for determining the contents of multiple components in the modified Qing'e pills according to claim 1, characterized in that: In the high performance liquid chromatography detection step, the chromatographic conditions also include: Column temperature 30-40°C, preferably 35°C; and / or, Flow rate 0.1-0.5 ml / min, preferably 0.3 ml / min; and / or, Detection wavelengths are 250nm and 350nm.

3. The method for determining the contents of multiple components in the modified Qing'e pills according to claim 1 or 2, characterized in that: The preparation method of the test solution comprises: taking a sample of the flavored Qing'e pills to be tested, adding an alcohol solution to extract, and collecting the extract to obtain the test solution.

4. The method for determining the contents of multiple components in the modified Qing'e pills according to claim 3, characterized in that: The preparation method of the test solution is: The alcohol solution comprises a methanol solution, preferably a 50-100 v / v% methanol solution; and / or, The extraction step comprises ultrasonic extraction, preferably the ultrasonic extraction power is 300-600W, and the ultrasonic extraction frequency is 20-60kHz; and / or, The extraction time of the extraction step is 15-45 min; and / or, The mass ratio of the sample to be tested of the flavored Qing'e Pills to the volume ratio of the alcohol solvent is (0.5-1.5):(25-50), wherein the mass unit of the sample to be tested of the flavored Qing'e Pills is g, and the volume unit of the alcohol solvent is ml.

5. The method for determining the contents of multiple components in the modified Qing'e pills according to claim 4, characterized in that: The method for preparing the test solution also includes the step of adding the flavored Qing'e pill to be tested sample into diatomaceous earth and mixing; Preferably, the mass ratio of the sample to be tested to the diatomaceous earth is 1-3:

1.

6. The method for determining the contents of multiple components in the modified Qing'e pills according to any one of claims 1 to 5, characterized in that: The detection indexes of the modified Qing'e pills include at least one of psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavones, psoralenine, psoralenidine, psoralen dihydroflavone methyl ether or isopsoralen chromene chalcone.

7. The method for determining the contents of multiple components in the modified Qing'e pills according to claim 6, characterized in that: The method also includes the step of performing linear equation fitting on the content detection of the detection index.

8. The method for determining the contents of multiple components in the modified Qing'e pills according to claim 7, characterized in that: In the modified Qing'e pills, the linear equations of different detection indicators include: Psoralen: y=20245x-4418.1; and / or, Isopsoralen: y=20661x-187.23; and / or, Psoralen: y=45454x-18464; and / or, Isopsoralen: y=48777x-15625; and / or, New psoralea corylifolia isoflavones: y=34253x-3344.6; and / or, Psoralen: y=60038x-2860; and / or, Psoralen: y=22522x-454.04; and / or, Psoralea corylifolia methyl ether: y=7643.8x-2270.4; and / or, Isopsoralea corylifolia chromone: y = 35692x-508.59; Where x is the injection concentration and y is the peak area.

9. Application of the method for determining the content of multiple components in the modified Qing'e pills according to any one of claims 1 to 8 in the field of quality inspection and control of the modified Qing'e pills.

10. A quality control method for flavored Qing'e pills, characterized in that: The method comprises the steps of determining the contents of multiple components in the modified Qing'e pills according to the method described in any one of claims 1 to 8; Preferably, the detection components of the modified Qing'e Pills include at least one of psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavones, psoralenine, psoralenidine, psoralen dihydroflavone methyl ether or isopsoralen chromene chalcone.

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