Quality standard detection method of zhenhuang submicroemulsion spray

Abstract

The present application relates to the technical field of drug quality standard detection, and particularly relates to a quality standard detection method of Zhenhuang submicroemulsion spray. The quality standard detection method comprises the following steps: adopting liquid chromatography-mass spectrometry to qualitatively detect gallic acid, radix scrophulariae glycoside and baicalin in the Zhenhuang submicroemulsion spray; adopting gas chromatography-mass spectrometry to qualitatively detect dextro-borneol in the Zhenhuang submicroemulsion spray; adopting liquid chromatography-mass spectrometry to quantitatively detect cholic acid in the Zhenhuang submicroemulsion spray; and adopting liquid chromatography-mass spectrometry to quantitatively detect bilirubin in the Zhenhuang submicroemulsion spray. The quality standard detection method is simple, convenient and accurate, can scientifically, normatively and effectively control the quality of the Zhenhuang submicroemulsion spray, and meets the requirements of drug safety, effectiveness and quality controllability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug quality standard detection, in particular to a quality standard detection method of precious yellow submicroemulsion spray. BACKGROUND

[0002] The precious yellow submicroemulsion spray is prepared from ox gall, pearl powder, acetic rhizoma sinapis, borneol, woodfordia, green fruit, medicinal oil phase, emulsifier, co-emulsifier, stabilizer and antioxidant, etc., and is a medicine capable of effectively preventing and treating radiotherapy-induced oropharyngeal mucositis.

[0003] The precious yellow submicroemulsion spray contains gallic acid, woodfordia glycoside, baicalin, dextro-borneol, cholic acid and bilirubin, etc., but there is no specific detection method for the quality standard of the precious yellow submicroemulsion spray in the prior art, resulting in non-standard quality of the spray. SUMMARY

[0004] The present application provides a quality standard detection method of the precious yellow submicroemulsion spray to solve the above technical problems in the prior art. The quality standard of the precious yellow submicroemulsion spray can scientifically, standardly and effectively control the quality of the precious yellow submicroemulsion spray, meet the requirements of drug safety, effectiveness and quality controllability, and determine how to detect the quality standard of the precious yellow submicroemulsion spray to be scientific and standard, and facilitate the operation of personnel in the industry.

[0005] According to the first aspect of the present application, the present application provides a quality standard detection method of the precious yellow submicroemulsion spray, comprising the following steps:

[0006] qualitatively determining gallic acid, woodfordia glycoside and baicalin in the precious yellow submicroemulsion spray by liquid chromatography-mass spectrometry (LC-MS);

[0007] qualitatively determining dextro-borneol in the precious yellow submicroemulsion spray by gas chromatography-mass spectrometry (GC-MS);

[0008] quantitatively determining cholic acid in the precious yellow submicroemulsion spray by LC-MS;

[0009] quantitatively determining bilirubin in the precious yellow submicroemulsion spray by LC-MS.

[0010] The quality standard detection method of the precious yellow submicroemulsion spray qualitatively determines gallic acid, woodfordia glycoside and baicalin in the precious yellow submicroemulsion spray, quantitatively determines cholic acid and bilirubin in the precious yellow submicroemulsion spray, and qualitatively determines dextro-borneol in the precious yellow submicroemulsion spray by GC-MS, so that the detection method is simple, convenient and accurate, the quality of the precious yellow submicroemulsion spray can be scientifically, standardly and effectively controlled, and the requirements of drug safety, effectiveness and quality controllability can be met.

[0011] Further, the qualitative determination of gallic acid, jianhu-diaomycin and baicalin in the Zhenhuang sub-micro emulsion spray by liquid chromatography-mass spectrometry specifically comprises the following steps:

[0012] A certain amount of methanol is measured into a centrifuge tube, and the Zhenhuang sub-micro emulsion is added and vortexed until completely dissolved. Then, a nylon membrane is used for filtration, and the filtrate is taken as the test solution one;

[0013] A certain amount of gallic acid, jianhu-diaomycin and baicalin reference substance is weighed, and methanol is added to prepare a reference solution one containing gallic acid, jianhu-diaomycin and baicalin;

[0014] A triple quadrupole liquid chromatography-mass spectrometer is used to test the test solution one and the reference solution one, respectively. The ion information of gallic acid, jianhu-diaomycin and baicalin in the test solution chromatogram is consistent with that in the reference solution chromatogram.

[0015] Further, in the preparation process of the test solution one, the volume ratio of methanol to Zhenhuang sub-micro emulsion is (15-20):1, preferably 19:1;

[0016] And / or, the concentration of gallic acid, jianhu-diaomycin and baicalin in the reference solution one is independently 80-120 ng / mL, preferably 100 ng / mL;

[0017] And / or, the chromatographic conditions for testing the test solution one and the reference solution one by the triple quadrupole liquid chromatography-mass spectrometer are as follows:

[0018] The chromatographic column is ACQUITY UPLC CSH, 2.1 mm x 100 mm, 1.7 μm; acetonitrile is used as mobile phase A, and 10 mM ammonium acetate 0.1% formic acid solution is used as mobile phase B for gradient elution; the column temperature is 35℃; the flow rate is 0.3 ml per minute; the injection volume is 2 μL;

[0019] The mass spectrometry system is equipped with ESI, and the analysis is carried out in positive and negative ion modes. The operating conditions are as follows: ESI+capillary voltage is 3000V, desolvation gas temperature is 350℃, ion source temperature is 150℃; ESI-capillary voltage is 3000V, desolvation gas temperature is 500℃, ion source temperature is 150℃; scanning mode: multiple reaction monitoring.

[0020] In the above scheme, by controlling the volume ratio of methanol to Zhenhuang sub-micro emulsion in the preparation process of the test solution, the extraction of gallic acid, rehmanniae radix glycosides and baicalin in Zhenhuang sub-micro emulsion is facilitated, thereby facilitating the accuracy of subsequent detection. By limiting the concentration of gallic acid, rehmanniae radix glycosides and baicalin in a suitable range, the accuracy of subsequent detection is facilitated. By limiting the chromatographic conditions for testing the test solution and the control solution, the content of gallic acid, rehmanniae radix glycosides and baicalin in Zhenhuang sub-micro emulsion can be accurately determined.

[0021] Further, the determination of dextro-borneol in Zhenhuang sub-micro emulsion by GC-MS includes the following steps:

[0022] A certain amount of methanol is measured in a centrifuge tube, and Zhenhuang sub-micro emulsion is added. After vortexing to completely dissolve, a nylon membrane is used for filtration, and the filtrate is taken as the test solution two;

[0023] A certain amount of natural borneol control sample is weighed, and methanol is added to prepare a control solution two of natural borneol;

[0024] The test solution two and the control solution two are tested by GC-MS, and the retention time of natural borneol in the test solution chromatogram is consistent with that in the control solution chromatogram.

[0025] Further, in the preparation process of the test solution two, the volume ratio of methanol to Zhenhuang sub-micro emulsion is (15-20):1, preferably 19:1;

[0026] And / or, the concentration of natural borneol in the control solution two is 80-120 μg / mL, preferably 100 μg / mL;

[0027] And / or, the chromatographic conditions for testing the test solution two and the control solution two by GC-MS are as follows:

[0028] Gas chromatography conditions: GsBP-5MS capillary column, 0.25 mm x 30 mm, 0.25 μm, initial temperature 80℃, heating rate 10℃ / min to 120℃, holding for 4 min; heating rate 20℃ / min to 240℃, holding for 5 min; carrier gas is high-purity He, flow rate is 1 mL / min, injection port temperature is 250℃, injection volume is 1 μL, split ratio is 20:1;

[0029] Mass spectrometry conditions: EI ion source; electron energy 70 eV; ion source temperature 230℃; MS quadrupole: 150℃; solvent cut-off time 2 min; mass scan range m / z 40-650; acquisition mode: full scan.

[0030] In the above scheme, by controlling the volume ratio of methanol to the precious yellow sub-micro emulsion in the preparation process of the test solution two, the extraction of dextro-borneol in the precious yellow sub-micro emulsion is facilitated, thereby facilitating the accuracy of the subsequent detection. By limiting the concentration of natural borneol in the control solution two within a suitable range, the accuracy of the subsequent detection is facilitated. By limiting the chromatographic conditions for testing the test solution two and the control solution two, the content of dextro-borneol in the precious yellow sub-micro emulsion spray can be accurately determined.

[0031] Further, the liquid chromatography-mass spectrometry is used to quantitatively determine the cholic acid component in the precious yellow sub-micro emulsion spray, which specifically comprises the following steps:

[0032] The methanol, dichloromethane and DMSO are uniformly mixed to form a solvent one;

[0033] The cholic acid control sample is weighed, methanol is added to prepare a control sample stock solution, and then an appropriate amount of the control sample stock solution is measured and diluted with the solvent one to obtain a control solution three;

[0034] The solvent one is measured and placed in a centrifugal tube, and the precious yellow sub-micro emulsion is added and vortexed to completely dissolve to obtain a test solution three;

[0035] The control solution three and the test solution three are respectively placed in centrifugal tubes and centrifuged, and the supernatant of the test solution three and the control solution three after centrifugation is tested by a triple quadrupole liquid chromatography-mass spectrometer, and the content of the cholic acid component is calculated according to the peak area by an external standard method.

[0036] In the process of determining the cholic acid in the sub-micro emulsion, two problems are faced: 1. A suitable sample pretreatment method needs to be established to fully extract the cholic acid in the sample to accurately determine the cholic acid in the sample. 2. A suitable liquid chromatography-mass spectrometry condition needs to be developed to accurately determine the cholic acid in the sample. According to the solubility of cholic acid, methanol can be selected as the extraction solvent of the sub-micro emulsion, but during the elution process of the test solution after extraction, the dissolved soybean oil will precipitate to a certain extent, which reduces the column efficiency of the chromatographic column. Therefore, in order to solve this drawback, the inventors used two pretreatment methods in the research: (1) adding a solvent component with stronger fat solubility to the cholic acid extraction solvent of the sub-micro emulsion to completely dissolve the oil phase of the emulsion. (2) destroying the emulsion to realize oil-water separation, and taking the sample for determination according to the solubility of the component to be determined. When the first pretreatment method is used, it is found that adjusting the solvent of the emulsion from methanol to a mixed solvent of methanol / dichloromethane / DMSO can more effectively dissolve various organic components in the emulsion, and the second pretreatment method uses anhydrous sodium sulfate to adsorb water and a 70°C heating method to destroy the emulsion, and the oil-water two phases are separated by centrifugation, and the water phase is taken for determination. The results have poor repeatability.

[0037] Further, the volume ratio of methanol, dichloromethane and DMSO in the solvent one is (0.5-1.5):(0.5-1.5):1, preferably 1:1:1;

[0038] And / or, the concentration of cholic acid in the control solution three is 1100-1200 ng / mL, preferably 1197 ng / mL;

[0039] And / or, during the preparation of the test solution three, the volume ratio of the solvent one to the precious yellow sub-micro emulsion is (15-20):1, preferably 19:1;

[0040] And / or, the chromatographic conditions for testing the supernatant of the test solution three and the control solution three after centrifugation by using a triple quadrupole liquid chromatograph-mass spectrometer are as follows:

[0041] The chromatographic column is ACQUITY UPLC CSH, 2.1 mm x 100 mm, 1.7 μm; acetonitrile is used as mobile phase A, and 10 mM ammonium acetate 0.1% formic acid solution is used as mobile phase B, gradient elution is performed according to the following table; the column temperature is 35°C; the flow rate is 0.3 ml per minute; the injection volume is 2 μL;

[0042] The mass spectrometry system is equipped with ESI, and the analysis is performed in negative ion mode, and the operating conditions are as follows: the capillary voltage is 3000 V, the desolvation gas temperature is 500°C, the ion source temperature is 150°C; the scanning mode is multiple reaction monitoring.

[0043] In the above scheme, by controlling the volume ratio of the solvent one to the precious yellow sub-micro emulsion during the preparation of the test solution three within a reasonable range, it is beneficial to the full extraction of cholic acid in the precious yellow sub-micro emulsion, thereby improving the accuracy of subsequent detection. By limiting the volume ratio of methanol, dichloromethane and DMSO in the solvent one within a suitable range, it is more beneficial to the full extraction of cholic acid in the precious yellow sub-micro emulsion, thereby improving the accuracy of subsequent detection. By limiting the concentration of cholic acid in the control solution three within a reasonable range, it is beneficial to the subsequent accurate comparison. Cholic acid is a substance with strong electronegativity, and after ESI ionization, the negative ion mode response is obvious, suitable for negative ion detection mode. Under the conditions of continuously optimizing the capillary voltage, desolvation gas temperature and ion source temperature, multiple reaction monitoring mode is adopted for quantitative determination of cholic acid. By limiting the chromatographic conditions for testing the test solution three and the control solution three, the content of cholic acid in the precious yellow sub-micro emulsion can be accurately determined.

[0044] Further, the quantitative determination of the bilirubin content in the precious yellow sub-micro emulsion by liquid chromatography-mass spectrometry includes the following steps:

[0045] Weigh methanol and DMSO, mix well to form solvent two;

[0046] Take the bilirubin control sample, first add dichloromethane to prepare the control sample stock solution, then take the control sample stock solution, add solvent two to dilute, and get the control sample solution four;

[0047] Take the solvent two into a centrifuge tube, add the Jinhuang sub-micro emulsion, and vortex to completely dissolve to get the test sample solution four;

[0048] Take the control sample solution four and the test sample solution four into a centrifuge tube respectively, centrifuge, and test the supernatant of the test sample solution four and the control sample solution four after centrifugation by using a triple quadrupole liquid chromatograph-mass spectrometer, and calculate the content of the bilirubin component according to the peak area by using an external standard method.

[0049] In the process of determining the bilirubin in the sub-micro emulsion, two problems are faced: 1. A suitable sample pretreatment method needs to be established to fully extract the bilirubin in the sample to accurately determine the bilirubin in the sample. 2. A suitable liquid quality condition needs to be developed to accurately determine the bilirubin in the sample. In the process of establishing a suitable sample pretreatment method, the inventors first used anhydrous sodium sulfate to adsorb moisture and a heating method to destroy the emulsion, centrifuged to separate the oil and water phases, and took the organic phase to determine. The results have poor repeatability. Therefore, the inventors seek a suitable solvent to extract the bilirubin in the sample. The inventors evaluated the solubility behavior of bilirubin in methanol and dichloromethane to prepare a suitable solvent for liquid quality analysis. The bilirubin control sample was dissolved in methanol, and bilirubin was found to precipitate, indicating that methanol is not a good solvent for bilirubin. The bilirubin control sample was dissolved in dichloromethane, and a clear bilirubin peak was detected, indicating that dichloromethane is a good solvent for bilirubin. However, bilirubin is easily retained in the sample injection needle, pipeline and chromatographic column, and there is still residue after multiple rinsing. The bilirubin control sample solution was continuously injected for 6 times, and the peak area RSD was greater than 15%, indicating poor stability. This is related to the strong volatility of dichloromethane, which can cause inaccurate quantification of bilirubin. Considering the polarity and volatility of methanol and dichloromethane, the solvent for the bilirubin control sample was optimized. Methanol and DMSO were used as mixed solvents to dilute the dichloromethane mother liquor of bilirubin, which met the solubility and made the solvent suitable for the liquid quality system. DMSO, as a universal solvent, can effectively dissolve various organic components in the emulsion, solve the problem of soybean oil precipitation in the test process, and overcome the damage of dichloromethane to the liquid quality system and the poor repeatability caused by strong volatility.

[0050] Further, the volume ratio of methanol and DMSO in the solvent two is (0.5-1.5):1, preferably 1:1;

[0051] And / or, the concentration of bilirubin in the control sample solution four is 100-110 ng / mL, preferably 105.1 ng / mL;

[0052] And / or, the volume ratio of solvent two to the precious yellow sub-micro emulsion in the preparation process of the test solution four is (15-20):1, preferably 19:1;

[0053] And / or, the chromatographic conditions for testing the supernatant of the test solution four and the control solution four after centrifugation by using the triple quadrupole liquid chromatograph-mass spectrometer are as follows:

[0054] The chromatographic column is ACQUITY UPLC CSH, 2.1 mm x 100 mm, 1.7 μm; methanol is used as the mobile phase A, and 10 mM ammonium acetate 0.1% formic acid solution is used as the mobile phase B, gradient elution is carried out according to the following table; the column temperature is 35°C; the flow rate is 0.3 ml per minute; the injection volume is 2 μL;

[0055] The mass spectrometry system is equipped with ESI, and the analysis is carried out in negative ion mode, and the operating conditions are as follows: the capillary voltage is 3000V, the desolvation gas temperature is 500°C, the ion source temperature is 150°C; the scanning mode is multiple reaction monitoring.

[0056] In the above scheme, by controlling the volume ratio of solvent two to the precious yellow sub-micro emulsion in the preparation process of the test solution four within a reasonable range, it is beneficial to the full extraction of bilirubin in the precious yellow sub-micro emulsion, thereby improving the accuracy of subsequent detection. By limiting the volume ratio of methanol and DMSO in solvent two within a suitable range, it is more beneficial to the full extraction of bilirubin in the precious yellow sub-micro emulsion, thereby improving the accuracy of subsequent detection. By limiting the concentration of bilirubin in the control solution four within a reasonable range, it is beneficial to the subsequent accurate comparison. Bilirubin is a substance with strong electronegativity, and after ESI ionization, the negative ion mode response is obvious, suitable for negative ion detection mode. Under the conditions of continuously optimizing the capillary voltage, desolvation gas temperature and ion source temperature, multiple reaction monitoring mode is adopted for quantitative determination of bilirubin. The solubility of bilirubin in different solvents is as follows: dichloromethane > DMSO > methanol > acetonitrile. The solubility of bilirubin in methanol is better than that in acetonitrile, and the elution effect of methanol is more ideal compared with that of the two mobile phases, so methanol is selected as the organic phase of the mobile phase. Gradient elution is carried out according to the program by using methanol as the mobile phase A and 10 mM ammonium acetate 0.1% formic acid solution as the mobile phase B. The gradient elution program takes into account the elution time and separation degree, and the effect is relatively ideal. By limiting the chromatographic conditions for testing the test solution four and the control solution four, the bilirubin component in the precious yellow sub-micro emulsion spray can be accurately determined.

[0057] The quality standard detection method of the precious yellow submicroemulsion spray provided by the application qualitatively determines the gallic acid, radix buddlejae glycosides and baicalin in the precious yellow submicroemulsion spray, quantitatively determines the cholic acid and bilirubin in the precious yellow submicroemulsion spray, and qualitatively determines the dextro-borneol in the precious yellow submicroemulsion spray by using gas chromatography-mass spectrometry. The detection method is simple, convenient and accurate, can scientifically, normatively and effectively control the quality of the precious yellow submicroemulsion spray, and meets the requirements of drug safety, effectiveness and quality controllability. BRIEF DESCRIPTION OF DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0059] Figure 1 The characteristic spectrum of gallic acid in the test sample solution one and the control sample solution one in the quality standard detection method of the precious yellow submicroemulsion spray provided by the application;

[0060] Figure 2 The characteristic spectrum of radix buddlejae glycosides in the test sample solution one and the control sample solution one in the quality standard detection method of the precious yellow submicroemulsion spray provided by the application;

[0061] Figure 3 The characteristic spectrum of baicalin in the test sample solution one and the control sample solution one in the quality standard detection method of the precious yellow submicroemulsion spray provided by the application;

[0062] Figure 4 The total ion current chromatogram of the control sample solution two and the test sample solution two in the quality standard detection method of the precious yellow submicroemulsion spray provided by the application;

[0063] Figure 5 The EI-MS spectrum of natural borneol in the control sample solution two and the test sample solution two in the quality standard detection method of the precious yellow submicroemulsion spray provided by the application;

[0064] Figure 6 The MRM chromatogram of the blank solution one, the control sample solution three and the test sample solution three in the quality standard detection method of the precious yellow submicroemulsion spray provided by the application;

[0065] Figure 7 The linear relationship diagram of the concentration and the peak area of the control sample solution three in the quality standard detection method of the precious yellow submicroemulsion spray provided by the application;

[0066] Figure 8The MRM chromatogram of the control solution three in the quality standard detection method of the Zhenhuang submicroemulsion spray provided by the application is as shown in the following figure when the concentration is 1.197 ng / ml.

[0067] Figure 9 The MRM chromatograms of the blank solution two, the control solution four and the test sample solution four in the quality standard detection method of the Zhenhuang submicroemulsion spray provided by the application are as shown in the following figures.

[0068] Figure 10 The linear relationship diagram of the concentration and the peak area of the control solution four in the quality standard detection method of the Zhenhuang submicroemulsion spray provided by the application is as shown in the following figure.

[0069] Figure 11 The MRM chromatogram of the control solution four in the quality standard detection method of the Zhenhuang submicroemulsion spray provided by the application is as shown in the following figure when the concentration is 0.4204 ng / ml. DETAILED DESCRIPTION

[0070] In order to make the purpose, technical scheme and advantages of the application more clear, the technical scheme in the application will be clearly and completely described below in combination with the drawings in the application. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0071] EMBODIMENT

[0072] A quality standard detection method of a Zhenhuang submicroemulsion spray, comprising the following steps:

[0073] I. Qualitative determination of gallic acid, woodfordatin and baicalin in the Zhenhuang submicroemulsion spray by using liquid chromatography-mass spectrometry.

[0074] 1. The instruments and reagents used are shown in the following table 1 and table 2.

[0075] Table 1

[0076] Instrument name Model Manufacturer Triple quadrupole liquid chromatography-mass spectrometer TQS Waters Electronic balance MS105 Mettler-Toledo Group, USA

[0077] Table 2

[0078] Reagent name Batch number Manufacturer Remarks Dichloromethane 20200305 Vake Chromatographically pure Methanol L6C0V45 Bioland Chromatographically pure DMSO LTB0V91 Bioland Chromatographically pure Acetonitrile 222721 Thermo Chromatographically pure Ammonium acetate 161197A Thermo Chromatographically pure Gallic acid 110831-201906 China Institute for Drug Control Reference substance 91.5% Oroxinol 111915-201804 China Institute for Drug Control Reference substance 90.4% Baicalin 110715-202223 China Institute for Drug Control Reference substance 97.2% Cholic acid 100078-201415 China Institute for Drug Control Reference substance 98.9% Bilirubin 100077-202009 China Institute for Drug Control Reference substance 98.8%

[0079] 2. Method and result

[0080] 2.1 Chromatographic conditions

[0081] Column: ACQUITY UPLC CSH (2.1 mm x 100 mm, 1.7 μm); mobile phase A: acetonitrile, mobile phase B: 10 mM ammonium acetate 0.1% formic acid solution, gradient elution was performed according to Table 3 below; column temperature was 35 °C; flow rate was 0.3 mL / min; injection volume was 2 μL.

[0082] The mass spectrometry system was a triple quadrupole, and the mass spectrometry conditions are shown in Table 4 below. An electrospray ionization source (ESI) was equipped, and analysis was performed in positive and negative ion modes, and the operating conditions were as follows: ①ESI+capillary voltage was 3000 V, desolvation gas temperature was 350 °C, ion source temperature: 150 °C. ②ESI- capillary voltage was 3000 V, desolvation gas temperature was 500 °C, ion source temperature: 150 °C. Scan mode: multiple reaction monitoring (MRM).

[0083] Table 3 Gradient table

[0084] Time (min) Mobile phase A (%) Mobile phase B (%) 0 20 80 8 80 20 8.1 20 80 10 20 80

[0085] Table 4 Mass spectrometry conditions

[0086] Compound + / - Parent ion Daughter ion Collision Oroxinol - 593.1 269.2 35 Baicalin + 447.0 271.1 25 Gallic acid - 169.0 125.1 15

[0087] 2.2 Control solution one

[0088] Accurately weigh 10.10 mg, 10.17 mg and 10.73 mg of gallic acid, oxymatrine and baicalin control substances respectively into 25 ml volumetric flasks, dissolve and dilute to the mark with methanol, shake well to obtain gallic acid (369.7 μg / mL), oxymatrine (367.7 μg / mL), and baicalin (417.2 μg / mL) control stock solutions respectively.

[0089] Accurately take an appropriate amount of each stock solution described above, dilute with methanol to obtain a mixed control solution one containing gallic acid, oxymatrine and baicalin at about 100 ng / mL.

[0090] 2.3 Test solution one

[0091] Accurately take 1900 μl of methanol into a centrifuge tube, add 100 μl of Zhenhuo submicron emulsion. After vortexing to completely dissolve, filter with a 0.22 μm nylon membrane, and take the filtrate for liquid phase determination.

[0092] 2.4 Results

[0093] The characteristic chromatograms of gallic acid, oxymatrine and baicalin in test solution one and control solution one are shown in the accompanying Figure 1-3 , wherein, Figure 1-3The left graph in each of the above figures is the control solution 1, and the right graph is the test solution 1. The ion peak retention time of gallic acid, plumbagin and baicalin in the test solution 1 and the control solution 1 is shown in Table 5.

[0094] As can be seen, the retention time of gallic acid, plumbagin and baicalin in the test solution 1 is consistent with that of the control solution 1, and the daughter ion information of the parent ions of gallic acid, plumbagin and baicalin in the test solution 1 is consistent.

[0095] Table 5 Ion peak retention time

[0096] Name Reference solution one (min) Test solution one (min) Gallic acid 0.87、1.09 0.87、1.09 Oroxinol 2.18 2.17 Baicalin 2.45 2.45

[0097] 3. Conclusion: The Zhenhuang sub-micro emulsion spray contains gallic acid, plumbagin and baicalin components. II. Qualitative determination of dextro-borneol component in Zhenhuang sub-micro emulsion spray by GC-MS.

[0098] 1. The instruments and reagents used are shown in Tables 6 and 7, respectively.

[0099] Table 6

[0100] Instrument name Model Manufacturer Gas chromatography-mass spectrometer Agilent 7890A / 5975C Agilent Electronic balance Mettler XS205 Mettler-Toledo Group, USA

[0101] Table 7

[0102]

[0103] 2. Method and results

[0104] 2.1 Chromatographic conditions

[0105] Gas chromatography conditions: GsBP-5MS capillary column (0.25 mm x 30 m, 0.25 μm), initial temperature 80℃, temperature increasing rate 10℃ / min to 120℃, holding for 4 min; temperature increasing rate 20℃ / min to 240℃, holding for 5 min; carrier gas high-purity He, flow rate 1 mL / min, injection port temperature 250℃, injection volume 1 μL, split ratio 20:1.

[0106] Mass spectrometry conditions: EI ion source; electron energy 70 eV; ion source temperature 230℃; MS quadrupole: 150℃; solvent cut-off time 2 min; mass scan range m / z 40-650; acquisition mode full scan.

[0107] 2.2 Control solution 2

[0108] Precisely weigh 15.40 mg of natural borneol control substance into a 25 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well to obtain a natural borneol (616.0 μg / mL) control substance stock solution.

[0109] Accurately take the above-mentioned stock solution into a centrifuge tube, dilute with methanol to obtain a control solution II containing about 100 μg / mL of natural borneol.

[0110] 2.3 Test solution II

[0111] Accurately take 1900 μl of methanol into a centrifuge tube, add 100 μl of Zhenhuo submicron emulsion. After ultrasonic dissolution, filter with a 0.22 μm nylon membrane, and take the filtrate for mass spectrometry.

[0112] 2.4 Results

[0113] Figure 4 Shown are the total ion chromatograms of control solution II and test solution II, wherein Figure 4 the upper graph corresponds to control solution II, and the lower graph corresponds to test solution II. It can be seen that the retention time of natural borneol in test solution II (5.54 min) is basically consistent with that of control solution II (5.52 min). Figure 5 Shown are the EI-MS spectra of natural borneol in control solution II and test solution II, wherein Figure 5 the upper graph corresponds to control solution II, and the lower graph corresponds to test solution II. It can be seen that the peak at 5.54 min in test solution II is searched in the EI-MS spectral library as dextro-borneol.

[0114] 2.5 It can be concluded from the above that Zhenhuo submicron emulsion contains dextro-borneol component.

[0115] III. Quantitative determination of cholic acid component in Zhenhuo submicron emulsion by liquid chromatography-mass spectrometry.

[0116] 1. The instruments and reagents used are shown in Tables 8 and 9, respectively.

[0117] Table 8

[0118] Instrument name Model Manufacturer Triple quadrupole liquid chromatography-mass spectrometer TQS Waters Electronic balance MS105 Mettler-Toledo Group, USA

[0119] Table 9

[0120] Reagent name Batch number Manufacturer Remarks Dichloromethane 20200305 Vake Chromatographically pure Methanol L6C0V45 Bioland Chromatographically pure DMSO LTB0V91 Bioland Chromatographically pure Acetonitrile 222721 Thermo Chromatographically pure Ammonium acetate 161197A Thermo Chromatographically pure Cholic acid 100078-201415 China Institute for Drug Control Reference substance 98.9%

[0121] 2. Experimental conditions

[0122] 2.1 Chromatographic conditions

[0123] Chromatographic column: ACQUITY UPLC CSH (2.1 mm x 100 mm, 1.7 μm); acetonitrile as mobile phase A, 10 mM ammonium acetate 0.1% formic acid solution as mobile phase B, gradient elution according to the following Table 10; column temperature 35°C; flow rate 0.3 ml / min; injection volume 2 μL.

[0124] The mass spectrometry system is a triple quadrupole, and the mass spectrometry conditions are shown in Table 11, equipped with an electrospray ionization source (ESI), and the analysis is performed in negative ion mode, and the operating conditions are as follows: capillary voltage is 3000 V, desolvation gas temperature is 500°C, ion source temperature: 150°C. Scan mode: multiple reaction monitoring (MRM). Table 10 Gradient table

[0125]

[0126]

[0127] Table 11 Mass spectrometry conditions

[0128] Compound + / - Parent ion Daughter ion Collision Cholic acid - 407.3 343.4 35

[0129] 2.2 Solution preparation

[0130] (1) Solvent one: take methanol: dichloromethane: DMSO = 1:1:1 (v:v:v) as a blank solution.

[0131] (2) Control solution three

[0132] Accurately weigh 12.10 mg of cholic acid control into a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, and obtain a control stock solution with a concentration of 239.35 μg / mL.

[0133] Accurately take the above stock solution, dilute with solvent to obtain a cholic acid control solution three with a concentration of 1197 ng / mL.

[0134] (3) Test solution three

[0135] Take 100 μl of Zhenhuo submicron emulsion and place it in a centrifuge tube, add 1900 μl of solvent one. Vortex to completely dissolve completely.

[0136] 2.3 Determination method

[0137] Take 1 ml of control solution three and test solution three respectively into a centrifuge tube, centrifuge at 10000 r / min for 5 min, take the supernatant for determination, and calculate the peak area by external standard method.

[0138] 3 Methodology verification

[0139] 3.1 Specificity

[0140] 3.1.1 Solution preparation

[0141] (1) Blank solution one: take solvent one as a blank solution.

[0142] (2) Control solution: prepare control solution three according to 2.2.

[0143] (3) Test solution: Prepare the test solution according to 2.2.

[0144] 3.1.2 Injection sequence: Take the above solution, and prepare according to the method of "2.3" to prepare, each injection 1 needle.

[0145] 3.1.3 Acceptable standard: The blank solution has no interference on the determination of cholic acid.

[0146] 3.1.4 Experimental results: As shown in Table 11, wherein, Figure 6 The upper graph of the blank solution shows the MRM chromatogram, the middle graph shows the MRM chromatogram of the reference solution three, and the lower graph shows the MRM chromatogram of the test solution three. As can be seen from Figure 6 , the blank solution has no interference at 2.211 min, the cholic acid peak in the reference solution three has a retention time of 2.211 min, and the cholic acid peak in the test solution three has a retention time of 2.211 min. Therefore, it can be concluded that the blank solution has no interference on the determination of cholic acid, and the method has good specificity. Figure 6

[0147] 3.2 Linear investigation

[0148] 3.2.1 Solution preparation

[0149] Take the reference stock solution under 2.2, and gradually dilute with solvent one to obtain a series of reference solution three.

[0150] 3.2.2 Injection sequence: Take the above solution, and prepare according to the method of "2.3" to prepare, each injection 1 needle.

[0151] 3.2.3 Acceptable standard: The correlation coefficient r of the regression curve is ≥0.990.

[0152] 3.2.4 Experimental results: As shown in Table 12. Figure 7

[0153] Table 12 Standard working curve

[0154]

[0155] 3.2.5 Conclusion

[0156] The linear relationship between the concentration of cholic acid and the peak area is good, the linear range is 239.3-2393 ng / ml, and the correlation coefficient r is 0.9987.

[0157] 3.3 Accuracy

[0158] 3.3.1 Solution preparation

[0159] ​​(1) Control solution: Take 0.5 ml of the control solution three with the concentration of 598.3 ng / ml prepared in 2.2, add 0.5 ml of the test solution three, mix well.

[0160] (2) Test solution: Prepare the test solution three according to 2.2.

[0161] (3) Low concentration investigation solution: Take 0.5 ml of the control solution three with the concentration of 598.3 ng / ml, add 0.5 ml of the test solution three, mix well.

[0162] (4) Medium concentration investigation solution: Take 0.5 ml of the control solution three with the concentration of 1197 ng / ml, add 0.5 ml of the test solution three, mix well.

[0163] (5) High concentration investigation solution: Take 0.5 ml of the control solution three with the concentration of 1795 ng / ml, add 0.5 ml of the test solution three, mix well. Prepare three parallel solutions for each concentration.

[0164] 3.3.2 Injection sequence: Take the above solutions, prepare according to the method in 2.3, and inject one needle each.

[0165] 3.3.3 Acceptable standard: The average recovery rate at each concentration should be 80-120%, and the RSD of the recovery rate of nine accuracy investigation solutions should be ≤10%.

[0166] 3.3.4 Experimental results: As shown in Table 13.

[0167] Table 13 Accuracy investigation results

[0168]

[0169] Calculation formula:

[0170]

[0171] 3.3.5 Conclusion

[0172] The average recovery rate of cholic acid is 102.0%, and the RSD is 3.5%, indicating that the method has good accuracy.

[0173] 3.4 Precision

[0174] 3.4.1 Solution preparation

[0175] Take the control solution three with the concentration of 598.3 ng / ml prepared in 2.2, and dilute it with solvent step by step.

[0176] 3.4.2 Injection sequence: Take the above solution, prepare according to the "determination method under item 2.3", and continuously inject 6 times.

[0177] 3.4.3 Acceptable criteria: RSD of peak area ≤10%.

[0178] 3.4.4 Experimental results: as shown in Table 14.

[0179] Table 14 Precision test results

[0180]

[0181] 3.4.5 Conclusion

[0182] The RSD of cholic acid peak area is 1.1% for 6 continuous injections, and the instrument precision is good.

[0183] 3.5 Reproducibility

[0184] 3.5.1 Solution preparation

[0185] (1) Test solution: prepared in parallel for 6 times according to the method under item 2.2.

[0186] 3.5.2 Injection sequence: Take the above solution, prepare according to the "determination method under item 2.3", and inject 1 time each.

[0187] 3.5.3 Acceptable criteria: RSD of peak area ≤10%.

[0188] 3.5.4 Experimental results: as shown in Table 15.

[0189] Table 15 Reproducibility test results

[0190]

[0191] 3.5.5 Conclusion

[0192] The RSD of cholic acid peak area is 1.4% for 6 reproducibility test solutions, and the method reproducibility is good.

[0193] 3.6 Stability

[0194] 3.6.1 Solution preparation

[0195] Stability test solution: accurately take 0.5 ml of 1197 ng / ml of the reference solution, add 0.5 ml of the test solution, and mix well.

[0196] 3.6.2 Injection sequence: Take the above solution, prepare according to the "determination method under item 2.3", and place it at 15°C, inject at 0, 1, 2, 4, 8 hours, and measure the peak area of cholic acid.

[0197] 3.6.3 Acceptance criteria: RSD of peak area ≤10% within 8 hours.

[0198] 3.6.4 Experimental results: as shown in Table 16.

[0199] Table 16 Stability investigation results

[0200]

[0201] 3.6.5 Conclusion

[0202] The RSD of the peak area of cholic acid in the stability investigation solution was 2.5% after being placed at 15°C for 8 hours, and the test sample spiked solution was stable in the solution.

[0203] 3.7 Limit of quantification and limit of detection

[0204] 3.7.1 Preparation of solutions

[0205] Limit of quantification investigation solution: the same as the control substance stock solution in item 2.2, which was diluted with solvent step by step to obtain a solution with a concentration of 2.393 ng / ml.

[0206] Limit of detection investigation solution: the same as the control substance stock solution in item 2.2, which was diluted with solvent step by step to obtain a solution with a concentration of 1.197 ng / ml.

[0207] 3.7.2 Injection sequence: take the above solutions, and prepare according to the “determination method in item 2.3” to prepare the limit of quantification investigation solution for 6 injections and the limit of detection investigation solution for 1 injection.

[0208] 3.7.3 Acceptable criteria: the peak area of the limit of quantification solution S / N ≥10, RSD ≤10%, and the peak area of the limit of detection solution S / N ≥3.

[0209] 3.7.4 Experimental results: as shown in Figure 6 and Table 17 below.

[0210] Table 17 Limit of quantification investigation results

[0211]

[0212] 3.7.5 Conclusion

[0213] (1) The limit of quantification concentration of cholic acid was 2.393 ng / ml. The peak area S / N of 6 determinations was ≥10, and the RSD of the peak area ratio was 7.9%.

[0214] (2) The limit of detection concentration of cholic acid was 1.197 ng / ml. The S / N of the limit of detection concentration solution was >3.

[0215] 3.8 Robustness

[0216] 3.8.1 Preparation of solutions

[0217] Reference solution: Take 3 of the reference solution under item 2.2.

[0218] 3.8.2 Investigated factors are as shown in Table 18 below

[0219] Table 18

[0220] Factor Standard condition Variation 1 Variation 2 Column temperature (℃) 35 33 37 Flow rate (ml / min) 0.30 0.28 0.32 Initial organic phase proportion (%) 20 18 22

[0221] 3.8.3 Injection sequence: Take the above solutions, prepare according to the "determination method under item 2.3", and inject 1 needle under each condition.

[0222] 3.8.4 Acceptable standard: RSD of peak area ≤10%.

[0223] 3.8.5 Experimental results: as shown in Table 19.

[0224] Table 19

[0225]

[0226] 3.8.6 Conclusion

[0227] When the flow rate, column temperature, initial mobile phase ratio and other conditions are slightly changed, the RSD of peak area is 4.6%, and the method is good in robustness.

[0228] 3.9 Summary

[0229] As shown in Table 20 below, by investigating the specificity, linearity, accuracy, precision, repeatability, stability, limit of quantification, limit of detection, and robustness of the cholic acid method, the results show that the method is accurate and reliable, and can be used for quantitative analysis of cholic acid in Zhenhuang submicron emulsion spray.

[0230] Table 20 Summary of the investigation of the cholic acid method

[0231]

[0232] 4. Sample determination

[0233] 4.1 Preparation of solutions

[0234] (1) Solvent I: Take methanol: dichloromethane: DMSO = 1:1:1 (v:v:v) as a blank solution.

[0235] (2) Reference solution three

[0236] Accurately weigh 12.10 mg of cholic acid reference substance into a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well to obtain a reference stock solution with a concentration of 239.35 μg / mL.

[0237] Accurately weigh about 100 mg of the above stock solution into a 10 ml volumetric flask, dilute with solvent to the mark, and mix to obtain a solution of the reference substance of cholic acid at a concentration of 1197 ng / mL.

[0238] (3) Test solution three

[0239] Take 100 μl of the Zhenhuo sub-micro emulsion into a centrifuge tube, add 1900 μl of solvent. Vortex to completely dissolve completely. Prepare 2 replicates in parallel.

[0240] 4.2 Determination method

[0241] Take 1 ml of the reference solution three and the test solution three respectively into a centrifuge tube, centrifuge at 10000 r / min for 5 min, take the supernatant to determine, and calculate the peak area by external standard method.

[0242] Injection sequence: Take the above solution, prepare according to the determination method under item 2.3, and inject 1 needle under each condition.

[0243] 4.3 Experimental results

[0244] As shown in Table 21.

[0245] Table 21 Determination results of samples

[0246]

[0247] Four, the liquid chromatography-mass spectrometry was used to quantitatively determine the bilirubin component in Zhenhuo sub-micro emulsion spray.

[0248] 1, The instruments and reagents used are shown in Tables 22 and 23 respectively.

[0249] Table 22

[0250] Instrument name Model Manufacturer Triple quadrupole liquid chromatography-mass spectrometer TQS Waters Electronic balance MS105 Mettler-Toledo Group, USA

[0251] Table 23

[0252] Reagent name Batch number Manufacturer Remarks Methanol L6C0V45 Bioland Chromatographically pure Dichloromethane 20200305 Vake Chromatographically pure DMSO LTB0V91 Bioland Chromatographically pure Ammonium acetate 161197A Thermo Chromatographically pure Bilirubin 100077-202009 China Institute for Drug Control Reference substance 98.8%

[0253] 2, Experimental conditions

[0254] 2.1 Chromatographic conditions

[0255] Chromatographic column: ACQUITY UPLC CSH (2.1 mm x 100 mm, 1.7 μm); methanol as mobile phase A, 10 mM ammonium acetate 0.1% formic acid solution as mobile phase B, gradient elution according to the following Table 24; column temperature 35 ℃; flow rate 0.3 ml per minute; injection volume 2 μL.

[0256] The mass spectrometry system was a triple quadrupole, and the mass spectrometry conditions are shown in Table 25, equipped with an electrospray ionization source (ESI), and analysis was performed in negative ion mode, with the following operating conditions: capillary voltage 3000 V, desolvation gas temperature 500 °C, ion source temperature: 150 °C. Scan mode: multiple reaction monitoring (MRM).

[0257] Table 24 Gradient table

[0258] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 4 100 0 6 100 0 6.1 60 40 10 60 40

[0259] Table 25 Mass spectrometry conditions

[0260] Compound + / - Parent ion Daughter ion Collision Bilirubin - 583.2 285.2 28

[0261] 2.2 Solution preparation

[0262] (1) Blank solution two: take methanol: DMSO = 1:1 (v:v) as a blank solution.

[0263] (2) Control solution four

[0264] Accurately weigh 10.64 mg of bilirubin control into a 100 ml volumetric flask, dissolve and dilute to the mark with dichloromethane, shake well to obtain a control stock solution with a concentration of 105.1 μg / mL. Accurately measure the above stock solution, dilute with solvent to obtain a bilirubin control solution with a concentration of 105.1 ng / mL.

[0265] (3) Test solution four

[0266] Take 100 μl of Zhenhu sub-microemulsion into a centrifuge tube, add 1900 μl of solvent. Vortex to completely dissolve.

[0267] 2.3 Determination method

[0268] Take 1 ml of control solution four and test solution four respectively into a centrifuge tube, centrifuge at 10000 r for 5 min, take the supernatant for determination, and calculate the peak area by external standard method.

[0269] 3, Methodology verification

[0270] 3.1 Specificity

[0271] 3.1.1 Solution preparation

[0272] (1) Blank solution two: take solvent two as a blank solution.

[0273] (2) Control solution: prepare control solution four according to 2.2.

[0274] (3) Test solution: prepare test solution four according to 2.2.

[0275] 3.1.2 Injection sequence: Take the above solution, according to the "determination method under item 2.3" prepared in accordance with the law, each injection 1 needle.

[0276] 3.1.3 Acceptable standard: Blank solution has no interference with the determination of bilirubin.

[0277] 3.1.4 Experimental results: As shown in Figure 9 , wherein, Figure 9 The upper graph corresponds to blank solution two, the middle graph corresponds to reference solution four, and the lower graph corresponds to test solution four. As can be seen from Figure 9 , blank solution two has no interference at 5.824 min, the reference solution four spectrum has a bilirubin peak retention time of 5.824 min, and the test solution four spectrum has a bilirubin peak retention time of 5.815 min. Therefore, it can be concluded that blank solution two has no interference with the determination of bilirubin, and the method is specific.

[0278] 3.2 Linear investigation

[0279] 3.2.1 Solution preparation

[0280] Take the reference stock solution under item 2.2, and gradually dilute it with solvent to obtain a series of reference solution four.

[0281] 3.2.2 Injection sequence: Take the above solution, according to the "determination method under item 2.3" prepared in accordance with the law, each injection 1 needle.

[0282] 3.2.3 Acceptable standard: The correlation coefficient r of the regression curve is ≥0.990.

[0283] 3.2.4 Experimental results: As shown in Figure 10 and Table 26.

[0284] Table 26 Standard working curve

[0285]

[0286] 3.2.5 Conclusion

[0287] The proposed method was used for detection, and the linear relationship between bilirubin concentration and peak area was good, the linear range was 21.02-210.2 ng / ml, and the correlation coefficient r was 0.9976.

[0288] 3.3 Accuracy

[0289] 3.3.1 Solution preparation

[0290] (1) Reference solution: Take the reference stock solution under item 2.2, and gradually dilute it with solvent to obtain reference solution four with concentrations of 52.56 ng / ml, 105.1 ng / ml and 157.7 ng / ml.

[0291] (2) Test solution: The test solution was prepared according to 2.2.

[0292] (3) Low concentration investigation solution: 0.5 ml of the control solution four at 52.56 ng / ml was precisely taken, 0.5 ml of the test solution four was added, and mixed.

[0293] (4) Medium concentration investigation solution: 0.5 ml of the control solution four at 105.1 ng / ml was precisely taken, 0.5 ml of the test solution four was added, and mixed.

[0294] (5) High concentration investigation solution: 0.5 ml of the control solution four at 157.7 ng / ml was precisely taken, 0.5 ml of the test solution four was added, and mixed. Three concentrations of solutions were prepared in triplicate.

[0295] 3.3.2 Injection sequence: The above solutions were prepared according to the determination method under item 2.3, and each was injected once.

[0296] 3.3.3 Acceptable standard: The average recovery rate at each concentration should be 80-120%, and the RSD of the recovery rate of nine accuracy investigation solutions should be ≤10%.

[0297] 3.3.4 Experimental results: As shown in Table 27.

[0298] Table 27 Accuracy investigation results

[0299]

[0300] Calculation formula:

[0301]

[0302] 3.3.5 Conclusion

[0303] The average recovery rate of bilirubin was 105.2%, and the RSD was 9.2%, indicating that the method had good accuracy.

[0304] 3.4 Precision

[0305] 3.4.1 Solution preparation

[0306] The control solution four at a concentration of 105.1 ng / ml was obtained by gradually diluting the control stock solution under item 2.2 with a solvent.

[0307] Injection sequence: The above solutions were prepared according to the determination method under item 2.3, and were continuously injected 6 times.

[0308] Acceptable standard: The RSD of peak area should be ≤10%.

[0309] Experimental results: As shown in Table 28.

[0310] Table 28 Precision Investigation Results

[0311]

[0312] 3.4.5 Conclusion

[0313] Six injections were continuously made, and the RSD of the bilirubin peak area was 6.3%, indicating that the precision of the instrument was good.

[0314] 3.5 Reproducibility

[0315] 3.5.1 Solution Preparation

[0316] Test solution: The test solution was prepared in the same manner as in item 2.2, and six injections were made in parallel.

[0317] 3.5.2 Injection Sequence: The above solution was prepared according to the method in item 2.3, and one injection was made each time.

[0318] 3.5.3 Acceptable Standard: The RSD of the peak area was ≤10%.

[0319] 3.5.4 Experimental Results: The results are shown in Table 29.

[0320] Table 29 Reproducibility Investigation Results

[0321]

[0322] 3.5.5 Conclusion

[0323] The RSD of the bilirubin peak area in the six reproducibility investigation solutions was 5.4%, indicating that the method was reproducible.

[0324] 3.6 Stability

[0325] 3.6.1 Solution Preparation

[0326] Stability investigation solution: 0.5 ml of the test solution was accurately measured from 105.1 ng / ml of the reference solution, and 0.5 ml of the test solution was added, and then mixed.

[0327] 3.6.2 Injection Sequence: The above solution was prepared according to the method in item 2.3, and one injection was made each time.

[0328] 3.6.3 Acceptable Standard: The RSD of the peak area was ≤10% within 10 hours.

[0329] 3.6.4 Experimental Results: The results are shown in Table 30.

[0330] Table 30 Stability Investigation Results

[0331]

[0332]

[0333] 3.6.5 Conclusion

[0334] The peak area RSD of bilirubin in the stability test solution was 4.4% after 10 hours of storage at 15°C, and the test sample spiked solution was stable in the solution.

[0335] 3.7 Limit of Quantification and Limit of Detection

[0336] 3.7.1 Solution Preparation

[0337] Limit of Quantification Test Solution: The control solution stock solution under item 2.2 was diluted with solvent step by step to obtain a solution with a concentration of 2.102 ng / ml.

[0338] Limit of Detection Test Solution: The control solution stock solution under item 2.2 was diluted with solvent step by step to obtain a solution with a concentration of 0.4204 ng / ml.

[0339] 3.7.2 Injection Sequence: The above solutions were prepared according to the "determination method under item 2.3" and the limit of quantification test solution was injected for 6 times. The limit of detection test solution was injected for 1 time.

[0340] 3.7.3 Acceptable Criteria: The peak area of the limit of quantification solution S / N≥10, RSD≤10%, and the peak area of the limit of detection solution S / N≥3.

[0341] 3.7.4 Experimental Results: As shown in Table 31. Figure 11

[0342] Table 31 Limit of Quantification Test Results

[0343]

[0344] 3.7.5 Conclusion

[0345] (1) The limit of quantification concentration of bilirubin was 2.102 ng / ml. The peak area S / N≥10 for 6 times, and the peak area ratio RSD=3.0%.

[0346] (2) The limit of detection concentration of bilirubin was 0.4204 ng / ml. The limit of detection concentration solution S / N>3.

[0347] 3.8 Robustness

[0348] 3.8.1 Solution Preparation

[0349] Control Solution: Four of the control solution under item 2.2 were taken.

[0350] 3.8.2 The test factors are shown in the following Table 32.​

[0351] Table 32

[0352] Factor Standard condition Variation 1 Variation 2 Column temperature (℃) 35 33 37 Flow rate (ml / min) 0.30 0.28 0.32 Initial organic phase proportion (%) 60 58 62

[0353] 3.8.3 Injection sequence: Take the above solution, and prepare according to the method in "2.3", inject 1 needle under each condition.

[0354] 3.8.4 Acceptable standard: RSD of peak area ≤10%.

[0355] 3.8.5 Experimental results: as shown in Table 33.

[0356] Table 33

[0357]

[0358] 3.8.6 Conclusion

[0359] When the flow rate, column temperature, initial mobile phase ratio and other conditions are slightly changed, the RSD of peak area is 6.8%, and the method is good in robustness.

[0360] 3.9 Summary

[0361] As shown in Table 34 below, by investigating the specificity, linearity, accuracy, precision, repeatability, stability, limit of quantification, limit of detection, and robustness of the bilirubin method, the results show that the method is accurate and reliable, and can be used for quantitative analysis of bilirubin in Zhenhuang submicron emulsion spray.

[0362] Table 34

[0363]

[0364]

[0365] 4. Sample determination

[0366] 4.1 Solution preparation

[0367] (1) Solvent two: take methanol: DMSO = 1:1 (v:v) as a blank solution.

[0368] (2) Control solution four

[0369] Accurately weigh 10.64 mg of bilirubin control into a 100 ml volumetric flask, dissolve and dilute to the mark with dichloromethane, shake well, and obtain a control stock solution with a concentration of 105.1 μg / mL. Accurately take the above stock solution, dilute with solvent to obtain a bilirubin control solution with a concentration of 105.1 ng / mL.

[0370] (3) Test solution four

[0371] Take 100 μl of Zhenhuang sub-micro emulsion into a centrifugal tube, and add 1900 μl of solvent. Vortex to completely dissolve. Prepare two samples in parallel.

[0372] 4.2 Determination method

[0373] Take 1 ml of control solution four and test solution four respectively into a centrifugal tube, centrifuge at 10000 r for 5 min, take the supernatant to determine, and calculate by peak area according to external standard method.

[0374] Injection sequence: take the above solution, prepare according to the determination method in item 2.3, and inject 1 needle under each condition.

[0375] 4.3 Experimental results

[0376] As shown in Table 35.

[0377] Table 35 Determination results of samples

[0378] Peak area Concentration (ng / ml) Content (μg / ml) Average content (ng / ml) Reference solution four 136538.344 105.1 Test solution four 1 33948.512 522.6 24.21 501.0 Test solution four 2 31118.846 479.2 24.08

[0379] The present application establishes a liquid quality method for qualitatively determining gallic acid, radix lonicerae japonicae and baicalin components in Zhenhuang sub-micro emulsion spray; and a gas quality method for qualitatively determining dextro-borneol component in Zhenhuang sub-micro emulsion spray. The results show that Zhenhuang sub-micro emulsion spray contains gallic acid, radix lonicerae japonicae, baicalin and dextro-borneol components.

[0380] The present application also establishes a liquid quality method for quantitatively determining cholic acid and bilirubin components in Zhenhuang sub-micro emulsion spray, and performs systematic methodological verification on the method. The results show that the established liquid quality method is accurate and reliable, and can be used for quantitative analysis of cholic acid and bilirubin in Zhenhuang sub-micro emulsion spray. The content of cholic acid in Zhenhuang sub-micro emulsion is 24.15 μg / ml, and the content of bilirubin is 501.0 ng / ml.

[0381] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting the quality standard of a yellow gold submicroemulsion spray, characterized in that, It comprises the following steps: Adopting liquid quality union to determine the qualitative determination of gallus acid, wood butterfly glycoside, Huangcen glycoside component in Zhenhuang submicro emulsion spray; Adopting gas quality union to determine the qualitative determination of right rotation dragon's blood component in Zhenhuang submicro emulsion spray; Adopting liquid quality union to determine the quantitative determination of gallus acid component in Zhenhuang submicro emulsion spray; Adopting liquid quality union to determine the quantitative determination of gallus acid component in Zhenhuang submicro emulsion spray; Adopting liquid quality union to determine the quantitative determination of gallus acid component in Zhenhuang submicro emulsion spray comprises the following steps: Mixing methanol, dichloromethane and DMSO uniformly to form solvent one; Weighing gallus acid control product, first adding methanol to prepare control product stock solution, then taking the control product stock solution to dilute with solvent one to obtain control product solution three; Weighing gallus acid control product, first adding methanol to prepare control product stock solution, then taking the control product stock solution to dilute with solvent one to obtain control product solution three; Taking the solvent one into centrifugal tube, adding Zhenhuang submicro emulsion, vortexing to completely dissolve to obtain test product solution three; Taking the control product solution three and test product solution three into centrifugal tube respectively, testing the supernatant of the control product solution three and test product solution three after centrifugation respectively by triple quadrupole liquid chromatography-mass spectrometry, and calculating the content of gallus acid component according to the peak area by external standard method; The quantitative determination of gallus acid component in Zhenhuang submicro emulsion spray by liquid quality union comprises the following steps: Weighing methanol and DMSO, mixing uniformly to form solvent two; Weighing gallus acid control product, first adding methanol to prepare control product stock solution, then taking the control product stock solution to dilute with solvent one to obtain control product solution three; Weighing gallus acid control product, first adding methanol to prepare control product stock solution, then taking the control product stock solution to dilute with solvent one to obtain control product solution three; 2. The quality standard detection method according to claim 1, characterized by, Taking the solvent one into centrifugal tube, adding Zhenhuang submicro emulsion, vortexing to completely dissolve to obtain test product solution three; Taking the control product solution three and test product solution three into centrifugal tube respectively, testing the supernatant of the control product solution three and test product solution three after centrifugation respectively by triple quadrupole liquid chromatography-mass spectrometry, and calculating the content of gallus acid component according to the peak area by external standard method; The quantitative determination of gallus acid component in Zhenhuang submicro emulsion spray by liquid quality union comprises the following steps: Weighing methanol and DMSO, mixing uniformly to form solvent two; 3. The quality standard detection method according to claim 2, characterized in that, Weighing gallus acid control product, first adding methanol to prepare control product stock solution, then taking the control product stock solution to dilute with solvent one to obtain control product solution three; Weighing gallus acid control product, first adding methanol to prepare control product stock solution, then taking the control product stock solution to dilute with solvent one to obtain control product solution three; Taking the solvent one into centrifugal tube, adding Zhenhuang submicro emulsion, vortexing to completely dissolve to obtain test product solution three; Taking the control product solution three and test product solution three into centrifugal tube respectively, testing the supernatant of the control product solution three and test product solution three after centrifugation respectively by triple quadrupole liquid chromatography-mass spectrometry, and calculating the content of gallus acid component according to the peak area by external standard method; The quantitative determination of gallus acid component in Zhenhuang submicro emulsion spray by liquid quality union comprises the following steps: Weighing methanol and DMSO, mixing uniformly to form solvent two; Weighing gallus acid control product, first adding methanol to prepare control product stock solution, then taking the control product stock solution to dilute with solvent one to obtain control product solution three; Weighing gallus acid control product, first adding methanol to prepare control product stock solution, then taking the control product stock solution to dilute with solvent one to obtain control product solution three; Taking the solvent one into centrifugal tube, adding Zhenhuang submicro emulsion, vortexing to completely dissolve to obtain test product solution three; Taking the control product solution three and test product solution three into centrifugal tube respectively, testing the supernatant of the control product solution three and test product solution three after centrifugation respectively by triple quadrupole liquid chromatography-mass spectrometry, and calculating the content of gallus acid component according to the peak area by external standard method; Chromatographic column: ACQUITY UPLC CSH, 2.1mm*100mm, 1.7μm; gradient elution was performed with acetonitrile as mobile phase A and 10mM ammonium acetate 0.1% formic acid solution as mobile phase B; the column temperature was 35℃; the flow rate was 0.3ml per minute; the injection volume was 2μL; The mass spectrometry system was equipped with ESI, and analysis was performed in positive and negative ion modes, and the operation conditions were as follows: ESI+capillary voltage was 3000V, desolvation gas temperature was 350℃, ion source temperature was 150℃; ESI-capillary voltage was 3000V, desolvation gas temperature was 500℃, ion source temperature was 150℃; scanning mode was multiple reaction monitoring.

4. The quality standard detection method according to claim 3, characterized in that, In the preparation process of the test solution, the volume ratio of methanol to Zhenhuang sub-micro emulsion was 19:

1.

5. The quality standard detection method according to claim 3, characterized in that, The concentration of gallic acid, radix platycodi glycosides and baicalin in the control solution one was independently 100ng / mL.

6. The quality standard detection method according to claim 1, characterized by, The qualitative determination of the dextro-borneol component in Zhenhuang sub-micro emulsion spray by GC-MS specifically includes the following steps: The methanol was measured and placed in a centrifuge tube, and Zhenhuang sub-micro emulsion was added. After vortexing to completely dissolve, it was filtered with a nylon membrane, and the filtrate was taken as the test solution two; The natural borneol control sample was weighed and added with methanol to prepare the control solution two of natural borneol; The GC-MS instrument was used to test the test solution two and the control solution two respectively, and the retention time of natural borneol in the test solution chromatogram was consistent with that in the control solution chromatogram.

7. The quality standard detection method according to claim 6, characterized in that, In the preparation process of the test solution two, the volume ratio of methanol to Zhenhuang sub-micro emulsion was (15-20):1; And / or, the concentration of natural borneol in the control solution two was 80-120μg / mL; And / or, the chromatographic conditions for testing the test solution two and the control solution two by the GC-MS instrument were as follows: Gas chromatography conditions: GsBP-5MS capillary column, 0.25mm*30mm, 0.25μm, initial temperature 80℃, temperature rising rate 10℃ / min to 120℃, holding for 4min; temperature rising rate 20℃ / min to 240℃, holding for 5min; carrier gas was high-purity He, flow rate was 1mL / min, injection port temperature was 250℃, injection amount was 1μL, split ratio was 20:1; Mass spectrometry conditions: EI ion source; electron energy 70eV; ion source temperature 230℃; MS quadrupole: 150℃; solvent cut-off time 2min; mass scan range m / z 40-650; acquisition mode: full scan.

8. The quality standard detection method according to claim 7, characterized in that, In the preparation process of the test solution two, the volume ratio of methanol to Zhenhuang sub-micro emulsion was 19:

1.

9. The quality standard detection method according to claim 7, characterized by, The concentration of natural borneol in the control solution two was 100μg / mL.

10. The quality standard detection method of claim 1, wherein, The volume ratio of methanol, dichloromethane and DMSO in the solvent one was (0.5-1.5):(0.5-1.5):1; And / or, the concentration of cholic acid in the control solution three was 1100-1200ng / mL; And / or, in the preparation process of the test solution three, the volume ratio of the solvent one to Zhenhuang sub-micro emulsion was (15-20):1; And / or, the triple quadrupole liquid chromatography-mass spectrometry instrument is used to test the supernatant of the test solution three and the control solution three after centrifugation, and the chromatographic conditions are as follows: The chromatographic column is ACQUITY UPLC CSH, 2.1mm*100mm, 1.7μm; the mobile phase A is acetonitrile, the mobile phase B is 10mM ammonium acetate 0.1% formic acid solution, and gradient elution is carried out according to the following table; the column temperature is 35°C; the flow rate is 0.3ml per minute; the injection volume is 2μL; The mass spectrometry analysis system is equipped with ESI, and the analysis is carried out in negative ion mode, and the operation conditions are as follows: the capillary voltage is 3000V, the desolvation gas temperature is 500°C, the ion source temperature is 150°C; the scanning mode is multiple reaction monitoring.

11. The quality standard detection method according to claim 10, characterized in that, The volume ratio of methanol, dichloromethane and DMSO in the solvent one is 1:1:

1.

12. The quality standard detection method of claim 10, wherein, The concentration of cholic acid in the control solution three is 1197ng / mL.

13. The quality standard detection method of claim 10, wherein, During the preparation of the test solution three, the volume ratio of solvent one to Zhenhuang submicron emulsion is 19:

1.

14. The quality standard detection method of claim 1, wherein, The volume ratio of methanol and DMSO in the solvent two is (0.5-1.5):1; And / or, the concentration of bilirubin in the control solution four is 100-110ng / mL; And / or, during the preparation of the test solution four, the volume ratio of solvent two to Zhenhuang submicron emulsion is (15-20):1; And / or, the triple quadrupole liquid chromatography-mass spectrometry instrument is used to test the supernatant of the test solution four and the control solution four after centrifugation, and the chromatographic conditions are as follows: The chromatographic column is ACQUITY UPLC CSH, 2.1mm*100mm, 1.7μm; the mobile phase A is acetonitrile, the mobile phase B is 10mM ammonium acetate 0.1% formic acid solution, and gradient elution is carried out according to the following table; the column temperature is 35°C; the flow rate is 0.3ml per minute; the injection volume is 2μL; The mass spectrometry analysis system is equipped with ESI, and the analysis is carried out in negative ion mode, and the operation conditions are as follows: the capillary voltage is 3000V, the desolvation gas temperature is 500°C, the ion source temperature is 150°C; the scanning mode is multiple reaction monitoring.

15. The quality standard detection method according to claim 14, characterized in that, The volume ratio of methanol and DMSO in the solvent two is 1:

1.

16. The quality standard detection method of claim 14, wherein, The concentration of bilirubin in the control solution four is 105.1ng / mL.

17. The method of claim 14, wherein the quality criterion is determined based on a comparison of the first and second values of the parameter. During the preparation of the test solution four, the volume ratio of solvent two to Zhenhuang submicron emulsion is 19:1.

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