Quality testing method for Hailong Gejie oral liquid

Abstract

The invention discloses a quality detection method for Hailong Gejie oral liquid, which belongs to the technical field of Chinese medicine component detection, and includes a method for measuring the content of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1 by high performance liquid chromatography-mass spectrometry and / or a method for measuring the content of baicalin and / or a thin layer chromatography identification method for Polygonum multiflorum. The quality detection method for Hailong Gejie oral liquid provided by the present invention measures the content of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1 in Hailong Gejie oral liquid by high performance liquid chromatography-mass spectrometry, measures the content of baicalin by high performance liquid chromatography, and identifies Polygonum multiflorum by thin layer chromatography; the detection method provided has good accuracy, stability and repeatability, and can more effectively and comprehensively detect, evaluate and control the quality of Hailong Gejie oral liquid, and ensure the quality and clinical effect of the product.

Description

Technical Field

[0001] The invention relates to a quality detection method for Hailong Geke oral liquid, belonging to the technical field of traditional Chinese medicine component detection. Background Art

[0002] Hailong Gejie oral liquid has the effects of warming the kidney and strengthening yang, replenishing essence and blood, and is clinically used for sore waist and feet, complexion White, impotence, spermatorrhea, uterine cold infertility, dizziness and other symptoms, the prescription is: Hailong 39.1g, Gecko 0.73g, Ginseng 4.68g, Sheep Whip 4.68g, Sheep Kidney 4.68g, Scutellaria 4.68g, Rehmannia Glutinosa 3.1g, Cuscuta 3.1g, Polygonum Multiflorum 3.1g, Rehmannia Glutinosa 3.1g, Tangerine Peel 3.1g, Angelica 1.56g, Astragalus 4.68g, Actinolite 1.56g, Lotus Stamen 1.56g, Licorice 1.56g, Chuanxiong 1.56g, Alisma Orientalis 1.56g, Cynomorium Songaricum 1.56g, Cardamom 1.56g, Agarwood 1.56g, Deer Antler 1.56g, Lycium Barbarum 1.56g, Meat 0.78g of Cistanche deserticola, 1.56g of roasted Epimedium, 1.56g of Cinnamon bark, 1.56g of Allium tuberosum, 1.56g of Cnidium monnieri, and 0.31g of Zanthoxylum bungeanum; preparation method: add water to the above 29 ingredients and boil twice, each time for 2 hours, combine the decoctions, concentrate to a clear paste with a relative density of 1.04-1.06 (80°C), add twice the amount of ethanol, refrigerate for 12 hours, filter, concentrate, add water, refrigerate, and filter; take 500g of sucrose, dissolve it in water, combine it with the above filtrate, mix well, add 1.7g of benzoic acid to dissolve it, adjust the pH value to 4.6-4.8 with sodium hydroxide, adjust the total amount to 1000ml, filter, package, and sterilize to obtain.

[0003] At present, the quality inspection of Hailong Gejie Oral Liquid mainly includes the identification of ginseng, scutellaria, tangerine peel, astragalus and the determination of the content of icariin. There are few inspection items, especially the identification method of ginseng in the prescription is thin layer chromatography, and the content of ginsenosides cannot be detected, which is not conducive to more effective control and inspection of the quality of the drug.

[0004] It should be noted that the above contents belong to the technical knowledge of the inventor and do not necessarily constitute prior art. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a quality detection method for Hailong Geke oral liquid, which can more effectively and comprehensively detect, evaluate and control the quality of Hailong Geke oral liquid, thereby ensuring the quality and clinical effect of the medicine.

[0006] The present invention achieves the above-mentioned purpose by adopting the following technical solutions:

[0007] The quality detection method of Hailong Gejie oral liquid includes a method for determining the contents of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1 by high performance liquid chromatography-mass spectrometry and / or a method for determining the content of baicalin and / or a method for thin layer chromatography identification of Polygonum multiflorum.

[0008] In a preferred embodiment, the method for determining the content of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1 by high performance liquid chromatography-mass spectrometry comprises the following steps:

[0009] (1) preparing a test solution and a mixed reference solution of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1;

[0010] (2) Determine chromatographic and mass spectrometric conditions;

[0011] The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler, column temperature at 40°C, flow rate at 0.3 ml / min, acetonitrile as mobile phase A, water as mobile phase B for gradient elution;

[0012] The mass spectrometry conditions were as follows: a triple quadrupole tandem mass spectrometer was used, the ion source was an electrospray ion source, the scanning mode was a negative ion mode, and the detection mode was multiple reaction monitoring;

[0013] (3) Determination: Take 1 μl of the test solution and the mixed reference solution respectively, inject them into the high performance liquid chromatography-mass spectrometer for determination, and calculate the content of each reference substance in the test solution based on the peak area.

[0014] Optionally, in step (2), the gradient elution program is: 0-12 min: 19% A, 81% B; 12-15 min: 19%→25% A, 81%→75% B; 15-35 min: 25%→80% A, 75%→20% B.

[0015] Optionally, in step (2), the mass spectrometry conditions further include:

[0016] The quantitative ion pair of ginsenoside Rg1 is m / z799.5→637.4, and the qualitative ion pair is m / z799.50→475.4;

[0017] The quantitative ion pair of ginsenoside Re is m / z945.5→637.4, and the qualitative ion pair is m / z945.55→475.4;

[0018] The quantitative ion pair of ginsenoside Rf is m / z799.5→637.4, and the qualitative ion pair is m / z799.50→475.4;

[0019] The quantitative ion pair of ginsenoside Rb1 was m / z1107.6→179.1, and the qualitative ion pair was m / z1107.6→945.6.

[0020] Optionally, in step (1), the preparation method of the test solution is as follows: take 100 ml of Hailong Gejie oral liquid, add 80 ml of ether, shake to extract, let stand, discard the ether solution, extract the water layer with water-saturated n-butanol for 3 times, 50 ml each time, combine the n-butanol solution, wash with ammonia test solution for 2 times, 50 ml each time, let stand for 12 hours after the first washing, combine the two ammonia test solutions, extract with water-saturated n-butanol for 2 times, 25 ml each time, combine the two n-butanol solutions, evaporate to dryness, add 2 ml of methanol to dissolve the residue, pass through a macroporous adsorption resin column, elute with 50 ml of water, 50 ml of 20% ethanol and 80 ml of 80% ethanol, collect the 80% ethanol eluate, evaporate to dryness, add methanol to dissolve the residue, and make the volume to 5 ml to prepare the test solution;

[0021] The preparation method of the mixed reference solution is as follows: take ginsenoside Rg1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rb1 reference substance respectively, add methanol to make a mixed reference solution containing 0.1 μg of each in 1 ml.

[0022] In a preferred embodiment, the method for determining the content of baicalin comprises the following steps:

[0023] (1) Preparation of test solution and baicalin reference solution:

[0024] The preparation method of the test solution is as follows: take 10 ml of Hailong Gejie oral liquid and place it in a 25 ml volumetric bottle, add methanol to dilute to the scale, shake well, filter, and take the filtrate to prepare the test solution;

[0025] The preparation method of baicalin reference solution is as follows: take 10.23 mg of baicalin reference, place it in a 250 ml volumetric bottle, add 60% methanol to dissolve and dilute to the scale, shake well, take 40 ml, place it in a 50 ml volumetric bottle, add 60% methanol to dilute to the scale, shake well, and prepare a baicalin reference solution with a concentration of 31.23 μg / ml;

[0026] (2) Determine the chromatographic conditions:

[0027] Octadecylsilane bonded silica was used as the filler, the column temperature was 25°C, the flow rate was 1 ml / min, the mobile phase was methanol-0.2% phosphoric acid solution with a volume ratio of 47:53, and the detection wavelength was 280 nm;

[0028] (3) Determination: Take 10 μl of baicalin reference solution and test solution respectively, inject them into liquid chromatography, and measure them. Calculate the baicalin content in the test solution based on the peak area.

[0029] In a preferred embodiment, the thin layer chromatography identification method of Polygonum multiflorum comprises the following steps:

[0030] (1) Prepare test sample solution, control medicinal material solution, rheum officinale reference solution, and negative control solution;

[0031] The preparation method of the test solution is as follows: take 50 ml of Hailong Gejie oral liquid, add 3 ml of 37% hydrochloric acid, add ethyl acetate and shake to extract 3 times, add 30 ml of ethyl acetate each time, combine the ethyl acetate extracts, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and prepare the test solution;

[0032] The preparation method of the control medicinal material solution is as follows: take 0.2 g of the control medicinal material of Polygonum multiflorum, add 0.5 ml of 37% hydrochloric acid, add 20 ml of ethyl acetate, ultrasonically treat for 20 minutes, filter, evaporate the filtrate, and concentrate the residue to 1 ml to prepare the control medicinal material solution;

[0033] The preparation method of the rheum officinale reference substance solution is as follows: 5.02 mg of the rheum officinale reference substance is taken, and 5 ml of methanol is added to dissolve it to prepare the rheum officinale reference substance solution;

[0034] Preparation of negative control solution: remove the other medicinal flavors of Polygonum multiflorum from the prescription of Hailong Gejie oral solution to prepare a negative control sample lacking Polygonum multiflorum, and use the same preparation method for the test solution to prepare a negative control solution;

[0035] (2) According to the thin layer chromatography test, 10-20 μl of the test sample solution, 2 μl of the control medicinal material solution, 1-2 μl of the rhein reference solution, and 10-20 μl of the negative control solution were respectively spotted on the same silica gel G thin layer plate, and the upper layer solution of a mixed solution of toluene-ethyl acetate-formic acid with a volume ratio of 15:2:1 was used as the developing solvent. The plate was developed for 8 cm, taken out, dried, and examined under an ultraviolet lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the control medicinal material and the rhein reference, and there were no spots at the corresponding positions of the negative control.

[0036] The beneficial effects of this application include but are not limited to:

[0037] The quality detection method of Hailong Gejie oral liquid provided by the invention adopts high performance liquid chromatography-mass spectrometry to determine the contents of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1 in the Hailong Gejie oral liquid, adopts high performance liquid chromatography to determine the content of baicalin, and adopts thin layer chromatography to identify Polygonum multiflorum. The detection method provided has good accuracy, stability and repeatability, and can more effectively and comprehensively detect, evaluate and control the quality of Hailong Gejie oral liquid, thereby ensuring the quality and clinical effect of the medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0039] Figure 1 This is the liquid chromatogram of ginseng (the peaks of the mixed reference substance from left to right are ginsenosides Rg1, Re, and Rb1);

[0040] Figure 2 This is the MRM mass spectrum of the ginsenoside mixed reference substance;

[0041] Figure 3 This is the MRM mass spectrum of Hailong Gecko Oral Liquid sample;

[0042] Figure 4 This is the MRM mass spectrum of the negative sample of Hailong Gejie oral liquid;

[0043] Figure 5 This is the linear relationship diagram for the determination of baicalin content;

[0044] Figure 6 This is the specific HPLC chromatogram for the determination of baicalin content;

[0045] Figure 7 This is the HPLC chromatogram of the durability of the chromatographic column of Hailong Gejie oral liquid;

[0046] Figure 8 TLC for identification of Polygonum multiflorum (temperature: 25°C, relative humidity: 50%; TLC plate: Merck, batch number: HX72325726; 1. Lack of Polygonum multiflorum negative control; 2. Sample 2203011; 3. Sample 2203012; 4. Sample 2203013; 5. Sample 2203014; 6. Sample 2203015; 7. Polygonum multiflorum reference medicinal material; 8. Rhein reference substance; 9. Sample 2203016; 11. Sample 2202015; 13. Sample 2006002; 10. Sample 2203017; 12. Sample 2011001);

[0047] Fig. 9Thin layer chromatogram for identification of Polygonum multiflorum (temperature: 25°C, relative humidity: 50%, Yantai Institute of Chemical Industry, batch number: 20210625; 1. Lack of Polygonum multiflorum negative control; 2. Sample 2203011; 3. Sample 2203012; 4. Sample 2203013; 5. Sample 2203014; 6. Sample 2203015; 7. Polygonum multiflorum reference medicinal material; 8. Rhein reference substance; 9. Sample 2203016; 10. Sample 2203017; 11. Sample 2202015; 12. Sample 2011001; 13. Sample 2006002);

[0048] Fig.10 Thin layer chromatogram for identification of Polygonum multiflorum (temperature: 4°C, relative humidity: 35%, Yantai Institute of Chemical Industry, batch number: 20210625; 1. Lack of Polygonum multiflorum negative control; 2. Sample 2203011; 3. Sample 2203012; 4. Sample 2203013; 5. Sample 2203014; 6. Sample 2203015; 7. Polygonum multiflorum reference medicinal material; 8. Rhein reference substance; 9. Sample 2203016; 10. Sample 2203017; 11. Sample 2202015; 12. Sample 2011001; 13. Sample 2006002);

[0049] Fig.11 This is a thin layer chromatogram of the sample spot amount investigation in the identification method of Polygonum multiflorum (temperature: 20°C, relative humidity: 45%, Yantai Institute of Chemical Industry, batch number: 20210625; 1. Polygonum multiflorum reference medicinal material; 2. Rhein reference substance; 3-7: sample 2203017, the sample spot amount from left to right is 5, 10, 15, 20, 25 μl. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail in the following content. However, it should be pointed out that the following specific embodiments only provide specific operation examples of the present invention in an exemplary manner, but the protection scope of the present invention is not limited thereto. The protection scope of the present invention is limited only by the claims. It is obvious to those skilled in the art that various other improvements and replacements can be made to the embodiments of the present invention within the protection scope defined by the claims of the present invention, and the same technical effects can still be achieved, thereby achieving the ultimate technical purpose of the present invention.

[0051] 1. Instruments and Materials:

[0052] 1.1 Instrument:

[0053] Shimadzu 8050 HPLC-MS / MS, AB SCIEX Triple Quad 6500 HPLC-MS / MS, Mettler Toledo XSE205 electronic balance.

[0054] 1.2 Materials:

[0055] Reference substances: Ginsenoside Rg1 reference substance (China National Institute for Food and Drug Control, batch number: 110703-202034), Ginsenoside Re reference substance (China National Institute for Food and Drug Control, batch number: 110754-202028), Ginsenoside Rf reference substance (China National Institute for Food and Drug Control, batch number: 111719-201806), Ginsenoside Rb1 reference substance (China National Institute for Food and Drug Control, batch number: 110704-202129).

[0056] Reagents: Acetonitrile and methanol were of chromatographic grade, water was purified water from Millipore, and other reagents were of analytical grade.

[0057] Hailong Gecko Oral Liquid is produced by Shandong Hongjitang Pharmaceutical Group Co., Ltd., sample batch numbers are 2203011, 2203012, 2203013, 2203014, 2203015, 2203016, 2203017, 2202015, 2011001, and 2006002.

[0058] 2. Determination of the content of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1

[0059] 2.1 Determination method:

[0060] In the present invention, high performance liquid chromatography-mass spectrometry is used to determine the contents of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1 in Hailong Gejie oral liquid, and the determination method comprises the following steps:

[0061] (1) preparing a test solution and a mixed reference solution of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1;

[0062] Among them, the preparation method of the test solution is: take 100ml of Hailong Geke oral liquid, add 80ml of ether, shake, let it stand, discard the ether solution, add water-saturated n-butanol to extract 3 times, 50ml each time, combine the n-butanol extracts, wash twice with ammonia test solution, 50ml each time, let the first washing stand for 12h, combine the two ammonia test solutions, backwash twice with water-saturated n-butanol, 25ml each time, combine the two n-butanol solutions, evaporate to dryness, add 2ml of methanol to the residue to dissolve it, pass it through a D101 macroporous adsorption resin column (inner diameter 1.5cm, column height 12cm), elute with 50ml of water, 50ml of 20% ethanol and 80ml of 80% ethanol in turn, collect the 80% ethanol eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution.

[0063] The preparation method of the mixed reference solution is as follows: take ginsenoside Rg1 reference substance, ginsenoside Re reference substance and ginsenoside Rb1 reference substance, add methanol to prepare a mixed reference solution containing 0.1 μg of each per 1 ml.

[0064] (2) Determine chromatographic and mass spectrometric conditions;

[0065] Chromatographic conditions:

[0066] Octadecylsilane bonded silica gel was used as the filler (chromatographic column inner diameter: 2.1 mm); acetonitrile was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the column temperature was 40° C.; and the flow rate was 0.3 ml / min.

[0067] Table 1 Gradient elution program

[0068]

[0069] Mass spectrometry conditions:

[0070] A triple quadrupole mass spectrometer was used in the electrospray ionization mode (ESI ﹣ ), multiple reaction monitoring (MRM), interface temperature: 300℃, desolvation temperature: 526℃, heating gas flow rate: 10L / min. The number of theoretical plates should be no less than 4000 based on ginsenoside Rg1.

[0071] The monitored ion pairs are shown in Table 2 below.

[0072] Table 2 Monitoring ion pairs

[0073] Monitoring ingredients Ion Pair CE(V) <![CDATA[Ginsenoside Rg1]]> 799.5→637.4* 26 799.50→475.4 38 Ginsenoside Re 945.5→637.4* 20 945.55→475.4 32 Ginsenoside Rf 799.5→637.4* 30 799.50→475.4 28 <![CDATA[Ginsenoside Rb1]]> 1107.6→179.1 34 1107.6→945.6* 36

[0074] * indicates quantitation ion pair.

[0075] (3) Determination: Take 1 μl of the test solution and the mixed reference solution respectively, inject them into the high performance liquid chromatography-mass spectrometer for determination, and calculate the content of each reference substance in the test solution based on the peak area.

[0076] Measurement results:

[0077] According to the method and conditions specified in 2.1, 10 batches of Hailong Geke oral liquid samples were tested. The results are shown in Table 3.

[0078] Table 3 Determination results of ginseng in Hailong Gejie oral liquid samples

[0079]

[0080] Each 1ml of Hailong Gejia oral liquid contains ginsenoside Rg1(C 42 H 72 O 14 ) shall not be less than 5ng / ml, ginsenoside Re(C 48 H 82 O 18 ) shall not be less than 5ng / ml, ginsenoside Rf(C 42 H 72 O 14 ) shall not be less than 5ng / ml, ginsenoside Rb1(C 54 H 92 O 23 ) shall not be less than 5ng / ml.

[0081] Furthermore, the applicant of the present invention also explored the liquid chromatogram of ginsenosides, and the specific method is as follows:

[0082] Preparation of mixed reference solution 1: Take ginsenoside Rg1 reference substance, ginsenoside Re reference substance and ginsenoside Rb1 reference substance and add methanol to prepare a mixed reference solution 1 containing 1 mg of each substance per 1 ml.

[0083] Take 1 μl of the test solution and mixed reference solution 1 in 2.1 and inject them into the liquid chromatograph respectively. Record the chromatograms. Figure 1 The liquid phase detection results showed that the test solution had no corresponding chromatographic peak at the same position as the mixed reference solution chromatographic retention time.

[0084] 2.2 Investigation of the preparation method of the test solution

[0085] 2.2.1 Whether to use ether for washing:

[0086] Sample 1: Take 100 ml of Hailong Gejie oral liquid sample and directly extract it with water-saturated n-butanol for 3 times. The subsequent steps are the same as

[0087] 2.1 Preparation method of test solution.

[0088] After testing, it was found that there was chromatographic peak interference in the detection of ginsenosides in sample 1, and it was determined that ether was needed to wash the test solution.

[0089] 2.2.2 Investigation of extraction solvent types:

[0090] Sample 2: Take 100ml of Hailong Gejie oral liquid, add 80ml of ether, shake to extract, let stand, discard the ether solution, extract the water layer with ethyl acetate 3 times, 50ml each time, and the subsequent steps are the same as the preparation method of the test solution in 2.1. The results show that ethyl acetate cannot be used for complete extraction, so it is determined to use n-butanol for extraction.

[0091] 2.2.3 Whether to use ammonia test solution for washing:

[0092] Sample 3: Take 100ml of Hailong Geke oral liquid, add 80ml of ether, shake to extract, let stand, discard the ether solution, extract the aqueous layer with water-saturated n-butanol for 3 times, 50ml each time, combine the n-butanol solutions, evaporate to dryness, add 2ml of methanol to the residue to dissolve it, pass it through a D101 macroporous adsorption resin column (inner diameter 1.5cm, column height 12cm), elute with 80ml of 80% ethanol, collect the eluate, evaporate to dryness, add methanol to the residue to dissolve it, and make up the volume to 5ml as sample 3.

[0093] Sample 4: Take 100ml of Hailong Gejie oral liquid, add 80ml of ether, shake to extract, place, discard the ether solution, extract the water layer with water-saturated n-butanol 3 times, 50ml each time, combine the n-butanol solution, wash once with 50ml ammonia test solution, place for 6h, extract twice with water-saturated n-butanol, 25ml each time, combine the two n-butanol solutions, evaporate to dryness, add 2ml of methanol to dissolve the residue, pass through D101 macroporous adsorption resin column (inner diameter 1.5cm, column height 12cm), elute with 80% ethanol 80ml, collect the eluate, evaporate to dryness, add methanol to dissolve the residue, and adjust the volume to 5ml as sample 4. After testing, the ginsenoside content detected in samples 3 and 4 was low, and it was determined that they were washed twice with 50ml ammonia test solution (the first time was placed for 12h).

[0094] 2.2.4 Whether to use D101 macroporous resin for purification:

[0095] Sample 5: Take 100ml of Hailong Gejie oral liquid, do not use D101 macroporous resin for purification, and the rest of the steps are the same as the preparation method of the test solution in 2.1. The results show that there is interference in the detection of ginsenosides in sample 5, and it is determined to use D101 macroporous resin for purification.

[0096] 2.3 Methodological investigation:

[0097] 2.3.1 Specificity test

[0098] Prepare the test solution and mixed reference solution according to the method in 2.1.

[0099] Preparation of negative control solution: Take the other medicinal ingredients in the prescription of Hailong Gejie oral liquid except ginseng, and prepare a negative control sample lacking ginseng according to the prescription ratio. Prepare the negative control solution in the same way as the preparation method of the test solution in 2.1.

[0100] Determination: Accurately pipette 5 μl of the test solution, mixed reference solution, and negative control solution respectively, inject into the HPLC-MS / MS instrument, and determine using the chromatographic and mass spectrometric conditions in 2.1. The results are shown in Figure 2-Figure 4 .

[0101] exist Figure 3 In the ion chromatogram of the test product shown, Figure 2 The chromatographic peaks of ginsenoside Rg1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rb1 mixed reference substance have consistent chromatographic retention times. Figure 4 There were no chromatographic peaks corresponding to the extracted ion current chromatograms of the reference substances in the negative control solution, indicating that the negative control had no interference and the method had good specificity.

[0102] 2.3.2 Investigation of linear relationship

[0103] Prepare reference solutions of different concentrations, determine the peak area according to the conditions in 2.1, plot the standard curve with the peak area integral as the ordinate (y) and the reference sample concentration as the abscissa (x). Each component shows a good linear relationship within its respective concentration range, as shown in Table 4.

[0104] Table 4 Linear relationship and linear range

[0105]

[0106] 2.3.3 Instrument precision test

[0107] Take the reference solution and repeat the injection 6 times according to the chromatographic mass spectrometry conditions in 2.1. The peak area RSDs of ginsenosides, Rg1, Re, Rf, and Rb1 were 2.5%, 3.6%, 3.0%, and 2.9%, respectively, indicating that the instrument has good precision.

[0108] 2.3.4 Repeatability test

[0109] Take Hailong Gejie oral liquid and prepare 6 test solutions in parallel according to the method in 2.1. Determine according to the chromatography-mass spectrometry conditions in 2.1. The RSDs of the contents of ginsenosides Rg1, Re, Rf, and Rb1 are 3.4%, 3.5%, 2.9%, and 3.9%, respectively, indicating that the method has good repeatability.

[0110] 2.3.5 Stability test

[0111] The same test solution was taken and measured according to the chromatographic mass spectrometry conditions in 2.1 at 0, 4, 8, 12, 16, and 24 hours. The RSDs of the peak areas of ginsenosides Rg1, Re, Rf, and Rb1 were 3.2%, 3.9%, 2.7%, and 3.3%, respectively, indicating that the sample had good stability within 24 hours.

[0112] 2.3.6 Sample recovery test

[0113] Accurately measure 50 ml of Hailong Gejie oral liquid, add ginsenoside Rg1, Re, Rf, Rb1 mixed reference solution relative to 100% of Hailong Gejie oral liquid, prepare according to the method in 2.1, and determine according to the chromatographic mass spectrometry conditions in 2.1, and calculate the recovery rate. The average recovery rates of ginsenoside Rg1, Re, Rf, and Rb1 are 89%, 82%, 86%, and 85%, respectively, which meet the analysis requirements.

[0114] 3. Determination of baicalin content:

[0115] 3.1 Determination method:

[0116] (1) Preparation of test solution and baicalin reference solution:

[0117] The preparation method of the test solution is as follows: take 10 ml of Hailong Gejie oral liquid and place it in a 25 ml volumetric bottle, add methanol to dilute to the scale, shake well, filter, and take the filtrate to prepare the test solution;

[0118] The preparation method of baicalin reference solution is as follows: take 10.23 mg of baicalin reference, place it in a 250 ml volumetric bottle, add 60% methanol to dissolve and dilute to the scale, shake well, take 40 ml, place it in a 50 ml volumetric bottle, add 60% methanol to dilute to the scale, shake well, and prepare a baicalin reference solution with a concentration of 31.23 μg / ml;

[0119] (2) Determine the chromatographic conditions:

[0120] Octadecylsilane bonded silica gel was used as the filler, the column temperature was 25°C, the flow rate was 1 ml / min, the mobile phase was methanol-0.2% phosphoric acid solution with a volume ratio of 47:53, the detection wavelength was 280 nm, and the theoretical plate number calculated based on the baicalin peak should be no less than 2000;

[0121] (3) Determination: Take 10 μl of baicalin reference solution and test solution respectively, inject them into liquid chromatography, and measure them. Calculate the baicalin content based on the peak area.

[0122] Measurement results:

[0123] According to the method and conditions in 3.1, 10 batches of Hailong Geke oral liquid samples were tested. The results are shown in Table 5.

[0124] Table 5 Baicalin content determination results

[0125]

[0126] 3.2 Methodological investigation

[0127] 3.2.1 Investigation of linear relationship

[0128] 1μl, 2μl, 5μl, 10μl, 15μl, and 20μl of the baicalin reference solution (31.23μg / ml) in 3.1 were accurately drawn and injected into the liquid chromatograph to determine the peak area integral value. With the reference sample injection amount as the horizontal axis and the peak area integral value as the vertical axis, a linear regression was performed to obtain the regression equation: y=3754.9x+323.47, with a correlation coefficient of r=1.0000. The test results showed that the baicalin injection amount had a good linear relationship with the peak area integral value when it was between 0.03123 and 0.62460μg. The measurement results are shown in Table 6, and the linear relationship is shown in Table 6. Figure 5 .

[0129] Table 6 Results of investigation on the linear relationship of baicalin

[0130]

[0131] 3.2.2 Instrument precision test

[0132] Accurately pipette 10 μl of baicalin reference solution (31.23 μg / ml) and inject it into the liquid chromatograph. Inject the sample 6 times continuously and measure the peak area integral value. The results are shown in Table 7.

[0133]

[0134] The test results show that the instrument has good precision.

[0135] 3.2.3 Sample stability test

[0136] Take Hailong Gejie Oral Liquid (Batch No.: 2203011) and prepare the test solution according to the preparation method of the test solution in 3.1. Inject once at a certain time, 10 μl each time, and measure the peak area integral value of baicalin. The observation is carried out for a total of 24 hours to observe the stability of the test components in the test solution during the detection process. The results are shown in Table 8.

[0137] Table 8 Stability test results

[0138]

[0139] The test results show that the test solution has good stability within 24 hours and can meet the measurement needs.

[0140] 3.2.4 Method repeatability study

[0141] Take Hailong Gejie Oral Liquid (Batch No.: 2203011) and prepare the test solution according to the preparation method of the test solution in 3.1, and prepare 6 copies in parallel.

[0142] Accurately pipette 10 μl of the reference solution in 3.1 and the test solution mentioned above, inject into the liquid chromatograph, measure and calculate. The results are shown in Table 9.

[0143] Table 9 Repeatability test results

[0144]

[0145] The test results showed that the repeatability of the content determination method was good.

[0146] 3.2.5 Sample recovery test

[0147] Preparation of baicalin reference solution: Accurately weigh 10.23 mg of baicalin reference, place in a 250 ml volumetric flask, accurately weigh, add 60% methanol to dissolve and dilute to the scale, shake well; then accurately measure 40 ml, place in a 50 ml volumetric flask, add 60% methanol to dilute to the scale, shake well, to obtain a baicalin reference solution with a concentration of 31.23 μg / ml.

[0148] Preparation of test solution: Take 5 ml of Hailong Geke oral solution with known content (batch number: 2203011, baicalin content is 0.0693 mg / ml), put it in a 25 ml volumetric flask, accurately add 10 ml of the above baicalin reference solution, add 60% methanol to dilute to the scale, shake well, filter, and take the filtrate to obtain, and prepare 6 copies in parallel.

[0149] Accurately pipette 10 μl of the baicalin reference solution and test solution respectively, inject into liquid chromatograph, measure and calculate. The results are shown in Table 10.

[0150] Table 10 Results of sample recovery investigation

[0151]

[0152] The test results showed that the recovery rate of the content determination method was good.

[0153] 3.2.6 Specificity test

[0154] Preparation of negative control solution: Take the other medicinal ingredients in the prescription of Hailong Gejie oral solution except for Scutellaria baicalensis, and prepare a negative control sample lacking Scutellaria baicalensis according to the prescription ratio. Prepare the negative control solution according to the preparation method of the test solution in 3.1.

[0155] Accurately pipette 10 μl of baicalin reference solution, test solution under repeatability test and negative control solution respectively, inject into liquid chromatograph for determination. The results are shown in Table 1. Figure 6 In the chromatogram of the test sample, there is a corresponding chromatographic peak at the same position as the chromatogram of the reference sample, while there is no corresponding chromatographic peak in the chromatogram of the negative control, indicating that other medicinal ingredients in the prescription have no interference with the determination results.

[0156] 3.2.7 Column durability test

[0157] Three different brands of chromatographic columns were used to test according to the chromatographic conditions in 3.1. The results showed that the chromatographic peaks of baicalin had good peak shapes and could achieve baseline separation with adjacent chromatographic peaks, indicating that the same type of chromatographic columns of different brands had no significant effect on the detection of baicalin, and the experimental conditions had a wide range of applicability.

[0158] The detailed information of the chromatographic column is shown in Table 11 and the chromatogram is shown in Figure 7 .

[0159] Table 11 Durability test results of different chromatographic columns

[0160]

[0161] 4. Thin layer chromatography identification method of Polygonum multiflorum:

[0162] 4.1 Identification method:

[0163] (1) Prepare test sample solution, control medicinal material solution, rheum officinale reference solution, and negative control solution;

[0164] The preparation method of the test solution is as follows: take 50 ml of Hailong Geke oral liquid, add 3 ml of 37% hydrochloric acid, add ethyl acetate and shake to extract 3 times, adding 30 ml of ethyl acetate each time, combine the ethyl acetate extracts, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and prepare the test solution; sample spot volume: 10-20 μl.

[0165] The preparation method of the control medicinal material solution is as follows: take 0.2g of Polygonum multiflorum control medicinal material, add 0.5ml of 37% hydrochloric acid, then add 20ml of ethyl acetate, ultrasonically treat for 20 minutes, filter, evaporate the filtrate, concentrate the residue to 1ml, and prepare the control medicinal material solution; sample volume: 2μl.

[0166] The preparation method of the rheum officinale reference substance solution is as follows: take 5.02 mg of the rheum officinale reference substance, add 5 ml of methanol to dissolve it, and prepare the rheum officinale reference substance solution; the sample spot volume is 1 to 2 μl.

[0167] Preparation of negative control solution: remove the other medicinal ingredients of Polygonum multiflorum from the prescription of Hailong Gejie oral solution to prepare a negative control sample lacking Polygonum multiflorum. Use the same preparation method for the test solution to prepare a negative control solution; sample volume: 10-20μl.

[0168] (2) According to the thin layer chromatography test, 10-20 μl of the test sample solution, 2 μl of the control medicinal material solution, 1-2 μl of the rhein reference solution, and 10-20 μl of the negative control solution were respectively spotted on the same silica gel G thin layer plate, and the upper layer solution of a mixed solution of toluene-ethyl acetate-formic acid in a volume ratio of 15:2:1 was used as the developing solvent. Develop 8 cm, take out, dry, and examine under ultraviolet light at 365 nm.

[0169] Results: In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the control medicinal materials and the rhubarb reference substance, while there were no spots at the corresponding positions of the negative control, indicating that the negative control had no interference.

[0170] 4.2 Method investigation:

[0171] The test was conducted using thin layer boards of different brands and different temperature and humidity conditions. See the thin layer photos for details. Figure 8-Figure 10 The spots in the chromatograms are clear and the separation effect is good.

[0172] The sample volume (5-25 μl) of the test solution was investigated and the results are shown in Fig.11 The final sample volume was determined to be 10-20 μl.

[0173] It should be noted that in the quality detection method provided by the present invention, the chromatographic conditions, mass spectrometry conditions and other measurement conditions are recommended conditions, and can be appropriately adjusted according to the specific conditions of the configured instrument in actual application. When the sample is injected into the detection instrument for measurement, a standard curve is drawn using a conventional method, and the content of the component to be measured is calculated based on the chromatographic peak area of ​​each component in the test solution and the established standard curve of each index component.

[0174] The above specific implementation manner cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any substitution, improvement or change made to the implementation manner of the present invention falls within the protection scope of the present invention.

[0175] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A quality testing method for Hailong Gejie oral liquid, characterized in that: It includes a method for determining the content of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1 by high performance liquid chromatography-mass spectrometry and / or a method for determining the content of baicalin and / or a method for thin layer chromatography identification of Polygonum multiflorum; The method for determining the content of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1 by high performance liquid chromatography-mass spectrometry comprises the following steps: (1) preparing a test solution and a mixed reference solution of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf and ginsenoside Rb1; The preparation method of the test solution is as follows: take 100 ml of Hailong Gejie oral liquid, add 80 ml of ether, shake to extract, place, discard the ether solution, extract the water layer with water-saturated n-butanol for 3 times, 50 ml each time, combine the n-butanol solution, wash with ammonia test solution for 2 times, 50 ml each time, place the first washing for 12 hours, combine the 2 ammonia test solutions, extract with water-saturated n-butanol for 2 times, 25 ml each time, combine the 2 n-butanol solutions, evaporate to dryness, add 2 ml of methanol to dissolve the residue, pass through a macroporous adsorption resin column, elute with 50 ml of water, 50 ml of 20% ethanol and 80 ml of 80% ethanol in turn, collect the 80% ethanol eluate, evaporate to dryness, add methanol to dissolve the residue, make the volume to 5 ml, and prepare the test solution; (2) Determine chromatographic and mass spectrometric conditions; The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler, column temperature at 40°C, flow rate at 0.3 ml / min, acetonitrile as mobile phase A, water as mobile phase B for gradient elution; The gradient elution program was: 0-12 min: 19% A, 81% B; 12-15 min: 19% → 25% A, 81% → 75% B; 15-35 min: 25% → 80% A, 75% → 20% B; The mass spectrometry conditions were as follows: a triple quadrupole tandem mass spectrometer was used, the ion source was an electrospray ion source, the scanning mode was a negative ion mode, and the detection mode was multiple reaction monitoring; (3) Determination: Take 1 μl of the test solution and the mixed reference solution respectively, inject them into the high performance liquid chromatography-mass spectrometer for determination, and calculate the content of each reference substance in the test solution based on the peak area; The method for determining the content of baicalin comprises the following steps: (1) Preparation of test solution and baicalin reference solution: The preparation method of the test solution is as follows: take 10 ml of Hailong Gejie oral liquid and place it in a 25 ml volumetric bottle, add methanol to dilute to the scale, shake well, filter, and take the filtrate to prepare the test solution; The preparation method of baicalin reference solution is as follows: take 10.23 mg of baicalin reference, place it in a 250 ml volumetric bottle, add 60% methanol to dissolve and dilute to the scale, shake well, take 40 ml, place it in a 50 ml volumetric bottle, add 60% methanol to dilute to the scale, shake well, and prepare a baicalin reference solution with a concentration of 31.23 μg / ml; (2) Determine the chromatographic conditions: Octadecylsilane bonded silica was used as the filler, the column temperature was 25°C, the flow rate was 1 ml / min, the mobile phase was methanol-0.2% phosphoric acid solution with a volume ratio of 47:53, and the detection wavelength was 280 nm; (3) Determination: Take 10 μl of baicalin reference solution and test solution respectively, inject into liquid chromatography, and measure, and calculate the baicalin content in the test solution based on the peak area; The thin layer chromatography identification method of Polygonum multiflorum comprises the following steps: (1) Prepare test sample solution, control medicinal material solution, rheum officinale reference solution, and negative control solution; The preparation method of the test solution is as follows: take 50 ml of Hailong Gejie oral liquid, add 3 ml of 37% hydrochloric acid, add ethyl acetate and shake to extract 3 times, add 30 ml of ethyl acetate each time, combine the ethyl acetate extracts, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and prepare the test solution; The preparation method of the control medicinal material solution is as follows: take 0.2 g of the control medicinal material of Polygonum multiflorum, add 0.5 ml of 37% hydrochloric acid, add 20 ml of ethyl acetate, ultrasonically treat for 20 minutes, filter, evaporate the filtrate, and concentrate the residue to 1 ml to prepare the control medicinal material solution; The preparation method of the rheum officinale reference substance solution is as follows: 5.02 mg of the rheum officinale reference substance is taken, and 5 ml of methanol is added to dissolve it to prepare the rheum officinale reference substance solution; Preparation of negative control solution: remove the other medicinal flavors of Polygonum multiflorum from the prescription of Hailong Gejie oral solution to prepare a negative control sample lacking Polygonum multiflorum, and use the same preparation method for the test solution to prepare a negative control solution; (2) According to the thin layer chromatography test, 10-20 μl of the test sample solution, 2 μl of the control medicinal material solution, 1-2 μl of the rhein reference solution, and 10-20 μl of the negative control solution were respectively spotted on the same silica gel G thin layer plate, and the upper layer solution of a mixed solution of toluene-ethyl acetate-formic acid with a volume ratio of 15:2:1 was used as the developing solvent. The plate was developed for 8 cm, taken out, dried, and examined under an ultraviolet lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the control medicinal material and the rhein reference, and there were no spots at the corresponding positions of the negative control.

2. The quality detection method of Hailong Gejie oral liquid according to claim 1, characterized in that, In step (2), the mass spectrometry conditions also include: The quantitative ion pair of ginsenoside Rg1 is m / z799.5→637.4, and the qualitative ion pair is m / z799.50→475.4; The quantitative ion pair of ginsenoside Re is m / z945.5→637.4, and the qualitative ion pair is m / z945.55→475.4; The quantitative ion pair of ginsenoside Rf is m / z799.5→637.4, and the qualitative ion pair is m / z799.50→475.4; The quantitative ion pair of ginsenoside Rb1 was m / z1107.6→179.1, and the qualitative ion pair was m / z1107.6→945.

6.

3. The quality detection method of Hailong Gejie oral liquid according to claim 1, characterized in that, In step (1), the preparation method of the mixed reference solution is: take ginsenoside Rg1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rb1 reference substance respectively, add methanol to make a mixed reference solution containing 0.1 μg of each of them per 1 ml.

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