A method for constructing a characteristic spectrum of Jinzhen oral liquid

Abstract

The present invention belongs to the technical field of traditional Chinese medicine analysis, and in particular relates to a method for constructing a characteristic spectrum of Jinzhen oral liquid. The present invention conducts methodological verification on the constructed characteristic spectrum method of Jinzhen oral liquid, including specificity, precision, repeatability, and stability experiments. In each experimental result, the RSD value of the relative retention time of each peak is ≤1.0%, and the RSD value of the relative peak area is ≤10.0%. This shows that the characteristic spectrum method is good and can reflect the major chemical components in Jinzhen oral liquid. The overall quality of Jinzhen oral liquid can be controlled more efficiently and quickly.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine analysis, and in particular relates to a method for constructing a characteristic spectrum of Jinzhen oral liquid. Background Art

[0002] Jinzhen Oral Liquid, derived from the folk pediatric prescription "Lingyang Qingfei San," clears heat and toxins, relieves phlegm and relieves coughs, and is primarily used for pediatric bronchitis. Jinzhen Oral Liquid, listed in the 2020 edition of the Chinese Pharmacopoeia (Volume 1), is a combination of eight traditional Chinese medicinal herbs: goat horn, scutellaria baicalensis, fritillaria cirrhosa, licorice, rhubarb, gypsum, artificial bezoar, and qingning stone. Studies of the characteristic spectrum of Jinzhen Oral Liquid in the literature have only identified one chromatographic peak, and there is currently no research on the characteristic spectrum of Jinzhen Oral Liquid in the patent.

[0003] Jinzhen Oral Liquid is composed of eight traditional Chinese medicines, each containing multiple chemical components (such as flavonoids, alkaloids, and saponins). Chromatographic peak overlap and interference between components are common, making separation difficult. Excipients in compound preparations (such as syrups and preservatives) can interfere with the extraction and purification of target ingredients, necessitating the development of efficient extraction methods. The solubility of different components varies significantly, making it difficult to achieve optimal extraction efficiency for all components using a single solvent system.

[0004] In view of this, the present invention is proposed to more comprehensively control its intrinsic quality. Summary of the Invention

[0005] In order to make up for the deficiencies in the prior art, the present invention provides a method for constructing a characteristic spectrum of Jinzhen oral liquid.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for constructing a characteristic spectrum of Jinzhen oral liquid, comprising the following steps:

[0008] (1) Preparation of reference solution: Take an appropriate amount of reference substance, weigh accurately, add methanol to make a solution containing 75 μg per 1 ml, shake well, and use it as the reference solution; the reference substances are: gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein;

[0009] (2) Preparation of test solution: Take 3 ml of Jinzhen oral liquid, add 3 ml of methanol-water-formic acid complex solvent with a volume ratio of 70:30:0.1, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, with ultrasonic parameters of power 200 W and frequency 40 kHz; centrifuge at 8000 rpm for 10 minutes, and take the supernatant; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, and balance the column with 5 ml of methanol and 5 ml of water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, wash impurities with 5 ml of water and 5 ml of 20% methanol in turn, and discard the effluent; use 5 ml The target component was eluted with 80% methanol containing 0.1% formic acid, and the eluate was collected. The eluate was concentrated under reduced pressure at 40°C to near dryness, and 1 ml of ethanol was added to dissolve the residue. 3 ml of 4°C pre-cooled deionized water was slowly added to the mixed solution of ethanol and the residue, and the mixture was allowed to stand at 4°C overnight. The mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected. The crystals were dissolved in 1 ml of methanol, vortexed, and filtered through a 0.22 μm organic filter membrane. The filtrate was used as the test solution.

[0010] (3) Determination: The reference solution and the test solution were injected into a high performance liquid chromatograph under the following chromatographic conditions, with 10 μl of each sample injected, and the chromatogram was recorded; the chromatographic conditions were: octadecylsilane bonded silica gel as the filler; acetonitrile-methanol with a volume ratio of 70:35 as the mobile phase A, and 0.05% phosphoric acid solution with 0.3 g of sodium dodecylsulfonate added per 100 ml as the mobile phase B, and elution was performed according to the specified gradient; the column temperature was 30°C; the flow rate was 1 ml per minute; the UV-Vis detector had a detection wavelength of 210-230 nm;

[0011] The elution gradient conditions were:

[0012] From 0 to 8 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 11:89;

[0013] From 8 to 9 minutes, the volume ratio of mobile phase A to phase B was changed from 11:89 to 20:80;

[0014] From 9 to 23 min, the volume ratio of mobile phase A:phase B changed from 20:80 to 21:79;

[0015] From 23 to 28 min, the volume ratio of mobile phase A:phase B changed from 21:79 to 35:65;

[0016] From 28 to 37 min, the volume ratio of mobile phase A:phase B changed from 35:65 to 37:63;

[0017] From 37 to 45 min, the volume ratio of mobile phase A:phase B changed from 37:63 to 45:55;

[0018] From 45 to 60 min, the volume ratio of mobile phase A:phase B was changed from 45:55 to 100:0;

[0019] From 60 to 63 min, the volume ratio of mobile phase A:phase B was changed from 100:0 to 5:95;

[0020] 63-75 min, mobile phase A:B, volume ratio 5:95;

[0021] (4) Generate a reference characteristic spectrum: Use the “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine” developed by the Chinese Pharmacopoeia Committee, select the chromatographic peaks that exist in the chromatograms of different batches of Jinzhen Oral Liquid as common peaks, and use the average value calculation method to generate a reference characteristic spectrum of Jinzhen Oral Liquid.

[0022] Preferably, the chromatographic column in step (3) is Topsil-C18 (4.6x250mm, 5um).

[0023] Preferably, the control characteristic spectrum generated in step (4) includes 11 chromatographic common peaks, specifically: peak 3 corresponding to gallic acid, peak 8 corresponding to baicalin, peak 9 corresponding to wogonin, peak 10 corresponding to ammonium glycyrrhizate, and peak 11 corresponding to rhein.

[0024] Preferably, the characteristic peak is based on the baicalin chromatographic peak as the reference peak, and the relative retention times of the 11 chromatographic peaks are as follows: peak 1 is 0.19, peak 2 is 0.22, peak 3 is 0.23, peak 4 is 0.36, peak 5 is 0.47, peak 6 is 0.61, peak 7 is 0.65, peak 9 is 1.11, peak 10 is 1.45, and peak 11 is 0.03.

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention established an HPLC characteristic spectrum for Jinzhen Oral Liquid, identified 11 characteristic peaks, and comprehensively demonstrated the chemical composition characteristics of Jinzhen Oral Liquid;

[0027] (2) The sample pretreatment of the present invention is simple and rapid. The sample extraction method was investigated, and the type and ratio of the mobile phase were investigated. Within the investigated range, the chromatographic method showed 11 characteristic peaks, and the relative retention time of each peak had a small change;

[0028] (3) The method of characteristic spectrum of Jinzhen oral liquid constructed in the present invention was methodologically verified, including specificity, precision, repeatability, and stability experiments. The RSD values ​​of the relative retention time of each peak in each experimental result were all ≤1.0%, and the RSD values ​​of the relative peak area were all ≤10.0%. This shows that the characteristic spectrum method is good and can reflect the major chemical components in Jinzhen oral liquid. It can more efficiently and quickly control the overall quality of Jinzhen oral liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below with reference to the accompanying drawings.

[0030] Figure 1 The chromatogram of the test sample extraction solvent was examined; among them, S1: methanol-water-formic acid solution with a volume ratio of 70:30:0.1; S2: methanol-water-formic acid solution with a volume ratio of 60:40:0.1; S3: methanol-water solution with a volume ratio of 70:30;

[0031] Figure 2 Wavelength inspection chromatogram; among them, S1: 210nm; S2: 230nm;

[0032] Figure 3 Specific HPLC chromatogram; where S1: negative; S2: baicalin control; S3: wogonin control; S4: gallic acid control; S5: rhein control; S6: ammonium glycyrrhizate control; S7: test sample: 230257;

[0033] Figure 4 Precision HPLC chromatogram; where S1-S6: precision 1-precision 6;

[0034] Figure 5 Repeatability HPLC chromatogram; where S1-S6: repeatability 1-repeatability 6;

[0035] Figure 6 Stability HPLC chromatogram; wherein, S1: 0h; S2: 2h; S3: 4h; S4: 8h; S5: 12h; S6: 24h;

[0036] Figure 7 HPLC chromatograms of eight batches of Jinzhen oral liquid test samples; among them, S1: 230257; S2: 230316; S3: 230411; S4: 230509; S5: 230612; S6: 230738; S7: 230921; S8: 231135;

[0037] Figure 8 Comparative characteristic spectrum of Jinzhen oral liquid. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the following examples and accompanying drawings. The instruments and equipment used in the examples are as follows:

[0039] 1. Instruments and reagents

[0040] 1.1 Instruments and Equipment

[0041] .

[0042] 1.2 Test materials

[0043] .

[0044] 1.3 Reference Material Information

[0045] .

[0046] 1.4 Sample Information

[0047] .

[0048] Example 1 Investigation of the method for constructing the characteristic spectrum of Jinzhen oral liquid

[0049] 1.1 Investigation of test sample preparation methods

[0050] 1.1.1 Investigation of test sample solvents

[0051] Chromatographic conditions

[0052] Chromatographic column: Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol (70:35), mobile phase B: 0.05% phosphoric acid solution (with 0.3 g of sodium dodecyl sulfate added per 100 ml), gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 230 nm. The number of theoretical plates calculated based on the baicalin peak should be no less than 5000.

[0053] .

[0054] Preparation of reference solution

[0055] Take appropriate amount of gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein reference substances, weigh accurately, add methanol to make a solution containing 75 μg per 1 ml, shake well, and use as reference solution.

[0056] Preparation of test solution Take 3 ml of Jinzhen oral liquid (batch number: 230257), add 3 ml of methanol-water-formic acid solution with a volume ratio of 70:30:0.1, methanol-water-formic acid solution with a volume ratio of 60:40:0.1, and methanol-water solution with a volume ratio of 70:30, respectively, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, and the ultrasonic parameters are power 200W and frequency 40kHz; centrifuge at 8000 rpm for 10 minutes, and take the supernatant; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, and balance the column with 5 ml of methanol and 5 ml of water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, wash impurities with 5 ml of water and 5 ml of 20% methanol in turn, and discard the effluent; use 5 ml The target component was eluted with 80% methanol containing 0.1% formic acid, and the eluate was collected; the eluate was concentrated under reduced pressure at 40°C to near dryness, and 1 ml of ethanol was added to dissolve the residue; 3 ml of 4°C pre-cooled deionized water was slowly added to the ethanol solution, and the mixture was allowed to stand at 4°C overnight. The mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected; the crystals were dissolved in 1 ml of methanol, vortexed and filtered through a 0.22 μm organic filter membrane, and the filtrate was used as the test solution.

[0057] Assay

[0058] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0059] like Figure 1 As shown in the figure, the solvent investigation results show that when the extraction solvent contains 0.1% formic acid and the extraction solvent volume ratio is 70:30:0.1, the peak separation is better and the peak shape is better. When the extraction solvent does not contain formic acid, the peak shape is poor and peaks are missing, which does not achieve the desired effect. Therefore, a methanol-water-formic acid solution with a volume ratio of 70:30:0.1 was selected as the extraction solvent.

[0060] Table 1 Results of relative retention time of solvent investigation

[0061] .

[0062] 1.2 Investigation of chromatographic conditions

[0063] 1.2.1 Wavelength Investigation

[0064] Chromatographic conditions

[0065] Chromatographic column: Topsil-C18 (4.6x250mm, 5µm); mobile phase A: acetonitrile-methanol (70:35), mobile phase B: 0.05% phosphoric acid solution (with 0.3g of sodium dodecyl sulfate added per 100ml), gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 210nm and 230nm. The number of theoretical plates calculated based on the baicalin peak should be no less than 5000.

[0066] .

[0067] Preparation of reference solution

[0068] Take appropriate amount of gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein reference substances, weigh accurately, add methanol to make a solution containing 75ug per 1ml, shake well, and use it as the reference solution.

[0069] Preparation of test solution

[0070] Take 3 ml of Jinzhen oral liquid, add 3 ml of methanol-water-formic acid complex solvent with a volume ratio of 70:30:0.1, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, with ultrasonic parameters of power 200 W and frequency 40 kHz; centrifuge at 8000 rpm for 10 minutes, and take the supernatant; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, and balance the column with 5 ml of methanol and 5 ml of water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, wash impurities with 5 ml of water and 5 ml of 20% methanol in turn, and discard the effluent; use 5 ml The target component was eluted with 80% methanol containing 0.1% formic acid, and the eluate was collected; the eluate was concentrated under reduced pressure at 40°C to near dryness, and 1 ml of ethanol was added to dissolve the residue; 3 ml of 4°C pre-cooled deionized water was slowly added to the ethanol solution, and the mixture was allowed to stand at 4°C overnight. The mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected; the crystals were dissolved in 1 ml of methanol, vortexed and filtered through a 0.22 μm organic filter membrane, and the filtrate was used as the test solution.

[0071] Assay

[0072] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0073] The results show that 11 characteristic peaks can be detected at wavelengths of 210nm~230nm, and the RSD of the relative retention time is less than 1.0%. Therefore, the wavelength of 210nm~230nm can meet the characteristic spectrum detection requirements. Figure 2 .

[0074] Table 2 Results of relative retention time of wavelength investigation

[0075] .

[0076] 1.2.2 Investigation of mobile phase types

[0077] Chromatographic Conditions 1: Column: Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol (70:35), mobile phase B: 0.05% phosphoric acid solution (with 0.3 g of sodium dodecyl sulfate added per 100 mL), gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 230 nm. The number of theoretical plates calculated based on the baicalin peak should be no less than 5000.

[0078] .

[0079] Chromatographic Conditions 2: Column: Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: 0.05% phosphoric acid solution (with 0.3 g of sodium dodecyl sulfate added per 100 mL), gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 230 nm. The number of theoretical plates calculated based on the baicalin peak should be no less than 5000.

[0080] .

[0081] Chromatographic Conditions 3: Column: Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol (70:35), mobile phase B: 0.05% phosphoric acid solution, gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 230 nm. The theoretical plate number calculated based on the baicalin peak should be no less than 5000.

[0082] .

[0083] Preparation of reference solution

[0084] Take appropriate amount of gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein reference substances, weigh accurately, add methanol to make a solution containing 75ug per 1ml, shake well, and use it as the reference solution.

[0085] Preparation of test solution

[0086] Take 3 ml of Jinzhen oral liquid, add 3 ml of methanol-water-formic acid complex solvent with a volume ratio of 70:30:0.1, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, with ultrasonic parameters of power 200 W and frequency 40 kHz; centrifuge at 8000 rpm for 10 minutes, and take the supernatant; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, and balance the column with 5 ml of methanol and 5 ml of water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, wash impurities with 5 ml of water and 5 ml of 20% methanol in turn, and discard the effluent; use 5 ml The target component was eluted with 80% methanol containing 0.1% formic acid, and the eluate was collected; the eluate was concentrated under reduced pressure at 40°C to near dryness, and 1 ml of ethanol was added to dissolve the residue; 3 ml of 4°C pre-cooled deionized water was slowly added to the ethanol solution, and the mixture was allowed to stand at 4°C overnight. The mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected; the crystals were dissolved in 1 ml of methanol, vortexed and filtered through a 0.22 μm organic filter membrane, and the filtrate was used as the test solution.

[0087] Assay

[0088] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0089] The results showed that when acetonitrile-methanol (70:35) was used as mobile phase A and 0.05% phosphoric acid solution (0.3 g of sodium dodecyl sulfate was added per 100 ml) was used as mobile phase B for elution, the chromatogram of the test sample presented a total of 11 characteristic peaks, with good peak separation and excellent peak shape; when acetonitrile was used as mobile phase A and 0.05% phosphoric acid solution (0.3 g of sodium dodecyl sulfate was added per 100 ml) as mobile phase B or acetonitrile-methanol (70:35) was used as mobile phase A and 0.05% phosphoric acid solution as mobile phase B for elution, all peaks were missing and the corresponding effect was not achieved.

[0090] Table 3 Relative retention time results of mobile phase type investigation

[0091] .

[0092] 1.2.3 Investigation of mobile phase ratio

[0093] Chromatographic conditions

[0094] Chromatographic column: Topsil C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol (70:35); mobile phase B: 0.05% phosphoric acid solution (with 0.3 g of sodium dodecyl sulfate added per 100 ml); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 230 nm. The number of theoretical plates calculated based on the baicalin peak should be no less than 5000.

[0095] Mobile phase ratio 1:

[0096] .

[0097] Mobile phase ratio 2:

[0098] .

[0099] Mobile phase ratio 3:

[0100] .

[0101] Preparation of reference solution

[0102] Take appropriate amount of gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein reference substances, weigh accurately, add methanol to make a solution containing 75ug per 1ml, shake well, and use it as the reference solution.

[0103] Preparation of test solution

[0104] Take 3 ml of Jinzhen oral liquid, add 3 ml of methanol-water-formic acid complex solvent with a volume ratio of 70:30:0.1, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, with ultrasonic parameters of power 200 W and frequency 40 kHz; centrifuge at 8000 rpm for 10 minutes, and take the supernatant; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, and balance the column with 5 ml of methanol and 5 ml of water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, and wash with 5 ml of water and 5 ml Wash impurities with 20% methanol and discard the effluent; elute the target component with 5 ml of 80% methanol containing 0.1% formic acid and collect the eluate; concentrate the eluate under reduced pressure at 40°C to near dryness, and add 1 ml of ethanol to dissolve the residue; slowly add 3 ml of 4°C pre-cooled deionized water to the ethanol solution, let it stand at 4°C overnight, centrifuge at 10,000 rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with 1 ml of methanol, vortex mix, and pass through a 0.22 μm organic filter membrane, and take the filtrate as the test solution.

[0105] Assay

[0106] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0107] The results showed that when eluted with a mobile phase ratio of 1, the test sample chromatogram showed a total of 11 characteristic peaks, with good peak separation and excellent peak shape; when eluted with a mobile phase ratio of 2 or a mobile phase ratio of 3, all peaks were missing and the corresponding effect was not achieved.

[0108] Table 4 Results of relative retention time of mobile phase ratio

[0109] .

[0110] Example 2: Methodological Verification of the Characteristic Spectrum of Jinzhen Oral Liquid

[0111] 2.1 Exclusivity

[0112] Chromatographic conditions

[0113] Chromatographic column: Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol (70:35); mobile phase B: 0.05% phosphoric acid solution (with 0.3 g of sodium dodecyl sulfate added per 100 ml); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 230 nm. The number of theoretical plates calculated based on the baicalin peak should be no less than 5000.

[0114] .

[0115] Preparation of reference solution

[0116] Take appropriate amount of gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein reference substances, weigh accurately, add methanol to make a solution containing 75ug per 1ml, shake well, and use it as the reference solution.

[0117] Preparation of test solution

[0118] Take 3 ml of Jinzhen oral liquid, add 3 ml of methanol-water-formic acid complex solvent with a volume ratio of 70:30:0.1, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, with ultrasonic parameters of power 200 W and frequency 40 kHz; centrifuge at 8000 rpm for 10 minutes, and take the supernatant; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, and balance the column with 5 ml of methanol and 5 ml of water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, wash impurities with 5 ml of water and 5 ml of 20% methanol in turn, and discard the effluent; use 5 ml The target component was eluted with 80% methanol containing 0.1% formic acid, and the eluate was collected; the eluate was concentrated under reduced pressure at 40°C to near dryness, and 1 ml of ethanol was added to dissolve the residue; 3 ml of 4°C pre-cooled deionized water was slowly added to the ethanol solution, and the mixture was allowed to stand at 4°C overnight. The mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected; the crystals were dissolved in 1 ml of methanol, vortexed and filtered through a 0.22 μm organic filter membrane, and the filtrate was used as the test solution.

[0119] Preparation of negative solution

[0120] Take an appropriate amount of excipients, add 3 ml of methanol-water-formic acid composite solvent with a volume ratio of 70:30:0.1, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, with ultrasonic parameters of power 200 W and frequency 40 kHz; centrifuge at 8000 rpm for 10 minutes, and take the supernatant; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, and balance the column with 5 ml of methanol and 5 ml of water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, wash impurities with 5 ml of water and 5 ml of 20% methanol in turn, and discard the effluent; use 5 ml The target component was eluted with 80% methanol containing 0.1% formic acid, and the eluate was collected; the eluate was concentrated under reduced pressure at 40°C to near dryness, and 1 ml of ethanol was added to dissolve the residue; 3 ml of 4°C pre-cooled deionized water was slowly added to the ethanol solution, and the mixture was allowed to stand at 4°C overnight. The mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected; the crystals were dissolved in 1 ml of methanol, vortexed and filtered through a 0.22 μm organic filter membrane, and the filtrate was used as the test solution.

[0121] Assay

[0122] Accurately pipette 10 μl of each of the negative solution, reference solution and test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0123] The results showed that the negative solution had no interference and good specificity. Figure 3 .

[0124] 2.2 Precision

[0125] Take 3 ml of Jinzhen oral solution (batch number: 230257) and prepare the test solution according to the test solution preparation method under "2.1". Inject 6 times according to the chromatographic conditions under "2.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0126] Table 5 Precision relative retention time results

[0127] .

[0128] Table 6 Precision relative peak area results

[0129] .

[0130] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 10.0%, indicating good precision. The chromatogram is attached. Figure 4 .

[0131] 2.3 Repeatability

[0132] Take 6 portions of 3 ml of Jinzhen Oral Liquid (Batch No.: 230257) and prepare the test solution according to the test solution preparation method under "2.1". Inject the sample according to the chromatographic conditions under "2.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0133] Table 7 Repeatability relative retention time results

[0134] .

[0135] Table 8 Repeatability relative peak area results

[0136] .

[0137] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 10.0%, with good repeatability. The chromatogram is attached. Figure 5 .

[0138] 2.4 Stability

[0139] Take 3 ml of Jinzhen oral solution (batch number: 230257) and prepare the test solution according to the test solution preparation method under "2.1". Inject the sample at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h according to the chromatographic conditions under "2.1". Record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0140] Table 9 Stability relative retention time results

[0141] .

[0142] Table 10 Stability relative peak area results

[0143] .

[0144] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 10.0%, indicating that the solution was stable. The chromatogram is attached. Figure 6 .

[0145] In summary, the characteristic spectrum has good specificity and there is no interference from negative samples, indicating that the characteristic detection method has strong specificity. In the precision test, the relative retention time RSDs are all less than 1.0%, and the relative peak area RSDs are all less than 10.0%, indicating good instrument precision. In the repeatability test, the relative retention time RSDs of each chromatographic peak are all less than 1.0%, and the relative peak area RSDs are all less than 10.0%, indicating good repeatability. In the stability test, the relative retention time RSDs of each chromatographic peak are all less than 1.0%, and the relative peak area RSDs are all less than 10.0%, indicating that the test solution is stable within 24 hours. This method has been well validated.

[0146] Example 3 Construction of the Control Characteristic Spectrum of Jinzhen Oral Liquid

[0147] Chromatographic conditions

[0148] Chromatographic column: Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol (70:35); mobile phase B: 0.05% phosphoric acid solution (with 0.3 g of sodium dodecyl sulfate added per 100 ml); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 230 nm. The number of theoretical plates calculated based on the baicalin peak should be no less than 5000.

[0149] .

[0150] Preparation of reference solution

[0151] Take appropriate amount of gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein reference substances, weigh accurately, add methanol to make a solution containing 75ug per 1ml, shake well, and use it as the reference solution.

[0152] Preparation of test solution

[0153] Take 3 ml of Jinzhen oral liquid, add 3 ml of methanol-water-formic acid complex solvent with a volume ratio of 70:30:0.1, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, with ultrasonic parameters of power 200 W and frequency 40 kHz; centrifuge at 8000 rpm for 10 minutes, and take the supernatant; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, and balance the column with 5 ml of methanol and 5 ml of water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, wash impurities with 5 ml of water and 5 ml of 20% methanol in turn, and discard the effluent; use 5 ml The target component was eluted with 80% methanol containing 0.1% formic acid, and the eluate was collected; the eluate was concentrated under reduced pressure at 40°C to near dryness, and 1 ml of ethanol was added to dissolve the residue; 3 ml of 4°C pre-cooled deionized water was slowly added to the ethanol solution, and the mixture was allowed to stand at 4°C overnight. The mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected; the crystals were dissolved in 1 ml of methanol, vortexed and filtered through a 0.22 μm organic filter membrane, and the filtrate was used as the test solution.

[0154] Assay

[0155] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0156] The chromatograms of the test samples of 8 batches of Jinzhen oral liquid were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and the chromatographic peaks present in the chromatograms of different batches of Jinzhen oral liquid were selected as common peaks; the average value calculation method was used to generate the reference characteristic spectrum, and the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in the attached Figures 7 and 8 .

[0157] Table 11 Relative retention time results of eight batches of Jinzhen oral liquid samples

[0158] .

[0159] Table 12 Relative peak area results of eight batches of Jinzhen oral liquid samples

[0160] .

[0161] The chromatograms of the eight batches of Jinzhen oral liquid samples all showed 11 common peaks. The RSDs of the relative retention times of the 11 common peaks were all less than 1.0%, and the RSDs of the relative peak areas were all less than 10.0%.

[0162] The chromatogram of the test sample showed 11 characteristic peaks, of which peaks 3, 8, 9, 10, and 11 corresponded to the peaks of the reference substance of gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein, respectively. The peak corresponding to the peak of the reference substance of baicalin was the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.19 (peak 1), 0.22 (peak 2), 0.23 (peak 3), 0.36 (peak 4), 0.47 (peak 5), 0.61 (peak 6), 0.65 (peak 7), 1.11 (peak 9), 1.45 (peak 10), and 0.03 (peak 11).

Claims

1. A method for constructing a characteristic spectrum of Jinzhen oral liquid, characterized in that: The following steps are involved: (1) Preparation of reference solution: accurately weigh the reference substance, add methanol to prepare a solution containing 75 μg per 1 ml, shake well, and use it as the reference solution; the reference substances are: gallic acid, baicalin, wogonin, ammonium glycyrrhizate, and rhein; (2) Preparation of test solution: Take Jinzhen oral solution, add methanol-water-formic acid composite solvent, and vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes; centrifuge for 10 minutes, and take the supernatant after centrifugation; add 0.1 mol / L ammonia water to the supernatant to adjust the pH to 7.0, let it stand for 10 minutes, centrifuge for 10 minutes, discard the precipitate, and retain the supernatant; use C18 solid phase extraction column to purify the sample, that is, purify the supernatant, and balance the column with 5 ml methanol and 5 ml water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, and balance it with 5 ml water, 5 ml Wash impurities with 20% methanol and discard the effluent; elute the target component with 5 ml of 80% methanol containing 0.1% formic acid and collect the eluate; concentrate the eluate under reduced pressure at 40°C to near dryness, then add ethanol to dissolve the residue; slowly add 4°C pre-cooled deionized water to the mixed solution of ethanol and the residue, let it stand at 4°C overnight, centrifuge at 10,000 rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with methanol, vortex mix, and pass through a 0.22 μm organic filter membrane, and take the filtrate as the test solution; (3) Determination: The reference solution and the test solution were injected into a high performance liquid chromatograph under the following chromatographic conditions, with 10 μl of each sample injected, and the chromatogram was recorded; the chromatographic conditions were: octadecylsilane bonded silica gel as the filler; acetonitrile-methanol with a volume ratio of 70:35 as the mobile phase A, and 0.05% phosphoric acid solution with 0.3 g of sodium dodecylsulfonate added per 100 ml as the mobile phase B, and elution was performed according to the specified gradient; the column temperature was 30°C; the flow rate was 1 ml per minute; the UV-visible detector had a detection wavelength of 210-230 nm; The elution gradient conditions were: From 0 to 8 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 11:89; From 8 to 9 minutes, the volume ratio of mobile phase A to phase B was changed from 11:89 to 20:80; From 9 to 23 min, the volume ratio of mobile phase A:phase B changed from 20:80 to 21:79; From 23 to 28 min, the volume ratio of mobile phase A:phase B changed from 21:79 to 35:65; From 28 to 37 min, the volume ratio of mobile phase A:phase B changed from 35:65 to 37:63; From 37 to 45 min, the volume ratio of mobile phase A:phase B changed from 37:63 to 45:55; From 45 to 60 min, the volume ratio of mobile phase A:phase B was changed from 45:55 to 100:0; From 60 to 63 min, the volume ratio of mobile phase A:phase B was changed from 100:0 to 5:95; 63-75 min, mobile phase A:B, volume ratio 5:95; (4) Generate a control characteristic spectrum: Select the chromatographic peaks that exist in the chromatograms of different batches of Jinzhen oral liquid as common peaks, and use the average value calculation method to generate a control characteristic spectrum of Jinzhen oral liquid.

2. The method for constructing a characteristic spectrum of Jinzhen oral liquid according to claim 1, characterized in that: In step (2), the mixed solution was ultrasonically treated for 15 minutes with ultrasonic parameters of 200 W power and 40 kHz frequency.

3. The method for constructing a characteristic spectrum of Jinzhen oral liquid according to claim 1, characterized in that: The centrifugal speed in step (2) is 8000 rpm.

4. The method for constructing a characteristic spectrum of Jinzhen oral liquid according to claim 1, characterized in that: The volume ratio of methanol-water-formic acid composite solvent added in step (2) is 70:30:0.1, and the volume is 3 ml.

5. The method for constructing a characteristic spectrum of Jinzhen oral liquid according to claim 1, characterized in that: The chromatographic column in step (3) is Topsil-C18 4.6x250mm, 5um.

6. The method for constructing a characteristic spectrum of Jinzhen oral liquid according to claim 1, characterized in that: The control characteristic spectrum generated in step (4) includes 11 common chromatographic peaks, specifically: Peak 3 corresponding to gallic acid, Peak 8 corresponding to baicalin, Peak 9 corresponding to wogonin, Peak 10 corresponding to ammonium glycyrrhizate, and Peak 11 corresponding to rhein; wherein the relative retention times of the 11 chromatographic peaks are as specified: Peak 1 is 0.19, Peak 2 is 0.22, Peak 3 is 0.23, Peak 4 is 0.36, Peak 5 is 0.47, Peak 6 is 0.61, Peak 7 is 0.65, Peak 9 is 1.11, Peak 10 is 1.45, and Peak 11 is 0.03.

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