Construction method of specific chromatogram of oral liquid of Jinvibration

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a construction method of a specific chromatogram of a Jinvibration oral liquid. According to the invention, methodology verification is carried out on the constructed specific chromatogram method of the Jinvibration oral liquid, and the methodology verification comprises specificity, precision, repeatability and stability experiments. In each experiment result, the RSD value of the relative retention time of each peak is less than or equal to 1.0%, and the RSD value of the relative peak area is less than or equal to 10.0%. The specific chromatogram method is good, and the method can reflect large types of chemical components in the Jinvibration oral liquid. And the overall quality of the Jinvibration 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 particularly relates to a method for constructing a characteristic chromatogram of Jinzhen Oral Liquid. Background Art

[0002] Jinzhen Oral Liquid is derived from the folk pediatric prescription "Lingyang Qingfei Powder" and has the effects of clearing heat and detoxifying, removing phlegm and relieving cough. It is mainly used for infantile bronchitis. Jinzhen Oral Liquid is included in the first volume of the Chinese Pharmacopoeia (2020 Edition) and is composed of 8 traditional Chinese medicines, namely Cornu Saigae Tataricae, Scutellaria baicalensis Georgi, Fritillaria thunbergii Miq. var. chekiangensis Hsiao et K. C. Hsia, Glycyrrhiza uralensis Fisch., Rheum palmatum L., Gypsum Fibrosum, Calculus Bovis Artifactus, and Lapis Chloriti. In the literature research on the characteristic chromatogram of Jinzhen Oral Liquid, only 1 chromatographic peak has been identified, and there is no research on the characteristic chromatogram of Jinzhen Oral Liquid in patents for the time being.

[0003] Jinzhen Oral Liquid is composed of 8 traditional Chinese medicines, and each medicinal material contains various types of chemical components (such as flavonoids, alkaloids, saponins, etc.). Chromatographic peak overlap or interference is likely to occur among the components, resulting in great separation difficulty. Excipients (such as syrup, preservatives) in compound preparations may interfere with the extraction and purification of target components, and it is necessary to develop an efficient extraction method. The solubility differences of different components are large, and a single solvent system is difficult to take into account the extraction efficiency of all components.

[0004] In view of this, the present invention is specifically proposed to more comprehensively control its internal 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 chromatogram of Jinzhen Oral Liquid.

[0006] The present invention is realized by the following technical solutions: The present invention provides a method for constructing a characteristic chromatogram of Jinzhen Oral Liquid, including the following steps: (1) Preparation of the reference substance solution: Weigh an appropriate amount of reference substances accurately, and dissolve them in methanol respectively to prepare solutions containing 75 μg per 1 ml, shake well, and use them as the reference substance solutions; the reference substances are: gallic acid, baicalin, wogonin, ammonium glycyrrhizinate, rhein. (2) Preparation of the test solution: Take 3 ml of Jinzhen Oral Liquid, add 3 ml of a methanol-water-formic acid composite solvent with a volume ratio of 70:30:0.1, and vortex for 1 minute; ultrasonically treat the mixture for 15 minutes with ultrasonic parameters of a power of 200 W and a frequency of 40 kHz; centrifuge at 8000 rpm for 10 minutes and take the supernatant; add 0.1 mol / L ammonia water dropwise to the supernatant to adjust the pH to 7.0, let stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; purify the sample using a C18 solid-phase extraction column, and equilibrate the column with 5 ml of methanol and 5 ml of water in sequence; load the centrifuged supernatant onto the column at a flow rate of 1 ml / min, wash the impurities with 5 ml of water and 5 ml of 20% methanol in sequence, 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 until nearly dry, add 1 ml of ethanol to dissolve the residue; slowly add 3 ml of deionized water pre-cooled to 4 °C to the mixed solution of ethanol and residue, let stand overnight at 4 °C, centrifuge at 10000 rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with 1 ml of methanol, vortex, and filter through a 0.22 μm organic filter membrane, and take the subsequent filtrate as the test solution; (3) Determination: Inject the reference solution and the test solution into a high-performance liquid chromatograph under the following chromatographic conditions, inject 10 μl each, and record the chromatogram; the chromatographic conditions are: using octadecylsilane-bonded silica gel as the filler; using acetonitrile-methanol with a volume ratio of 70:35 as mobile phase A, and using a 0.05% phosphoric acid solution containing 0.3 g of sodium dodecyl sulfate per 100 ml as mobile phase B, and perform elution according to the specified gradient; the column temperature is 30 °C; the flow rate is 1 ml per minute; use an ultraviolet-visible (UV-Vis) detector, and the detection wavelength is 210 - 230 nm; The elution gradient conditions are: 0 - 8 min, mobile phase A: mobile phase B, volume ratio changes from 5:95 to 11:89; 8 - 9 min, mobile phase A: mobile phase B, volume ratio changes from 11:89 to 20:80; 9 - 23 min, mobile phase A: mobile phase B, volume ratio changes from 20:80 to 21:79; 23 - 28 min, mobile phase A: mobile phase B, volume ratio changes from 21:79 to 35:65; 28 - 37 min, mobile phase A: mobile phase B, volume ratio changes from 35:65 to 37:63; 37 - 45 min, mobile phase A: mobile phase B, volume ratio changes from 37:63 to 45:55; 45 - 60 min, mobile phase A: mobile phase B, volume ratio changes from 45:55 to 100:0; From 60 to 63 min, mobile phase A: B, the volume ratio changes from 100:0 to 5:95; From 63 to 75 min, mobile phase A: B, the volume ratio is 5:95; (4) Generate a control characteristic chromatogram: Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" formulated by the Pharmacopoeia Commission of the People's Republic of China, select the chromatographic peaks that exist in the chromatograms of different batches of Jinzhen Oral Liquid as common peaks, and generate the control characteristic chromatogram of Jinzhen Oral Liquid by the average value calculation method.

[0007] Preferably, in step (3), the chromatographic column is Topsil-C18 (4.6x250mm, 5um).

[0008] Preferably, the control characteristic chromatogram 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 glycyrrhizinate, and peak 11 corresponding to rhein.

[0009] Preferably, taking the baicalin chromatographic peak as the reference peak, the relative retention times of the 11 chromatographic peaks are specified values: 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.

[0010] Advantages of the present invention: (1) The present invention has established an HPLC characteristic chromatogram for Jinzhen Oral Liquid, identified 11 characteristic peaks, and more fully and comprehensively demonstrated the chemical composition characteristics of Jinzhen Oral Liquid; (2) The sample pretreatment of the present invention is simple and rapid. The extraction method of the sample is investigated, and the types and proportions of the mobile phase are investigated. Within the investigated range, 11 characteristic peaks are presented in this chromatographic method, and the relative retention times of each peak change little; (3) The present invention has carried out methodological verification on the established characteristic chromatogram method of Jinzhen Oral Liquid, including specificity, precision, repeatability, and stability experiments. In the results of each experiment, the RSD values of the relative retention times of each peak are ≤1.0%, and the RSD values of the relative peak areas are ≤10.0%. It shows that this characteristic chromatogram method is good, and this method can reflect the major chemical components in Jinzhen Oral Liquid. It can more efficiently and quickly control the overall quality of Jinzhen Oral Liquid. Description of the Drawings

[0011] The present invention will be further described below with reference to the drawings.

[0012] Figure 1Chromatogram of the extraction solvent of the test sample; 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; Figure 2 Chromatogram of wavelength investigation; among them, S1: 210 nm; S2: 230 nm; Figure 3 Specificity HPLC chromatogram; among them, S1: negative; S2: baicalin control; S3: wogonin control; S4: gallic acid control; S5: rhein control; S6: ammonium glycyrrhizinate control; S7: test sample: 230257; Figure 4 Precision HPLC chromatogram; among them, S1-S6: precision 1-precision 6; Figure 5 Repeatability HPLC chromatogram; among them, S1-S6: repeatability 1-repeatability 6; Figure 6 Stability HPLC chromatogram; among them, S1: 0 h; S2: 2 h; S3: 4 h; S4: 8 h; S5: 12 h; S6: 24 h; Figure 7 HPLC chromatograms of eight batches of test samples of Jinzhen Oral Liquid; among them, S1: 230257; S2: 230316; S3: 230411; S4: 230509; S5: 230612; S6: 230738; S7: 230921; S8: 231135; Figure 8 Characteristic fingerprint of Jinzhen Oral Liquid. Detailed implementation manners

[0013] The present invention will be further described below in conjunction with the embodiments and the accompanying drawings. The instruments and equipment used in the embodiments are as follows: I. Instruments and test drugs 1.1 Instrument and equipment 。

[0014] 1.2 Test materials 。

[0015] 1.3 Information of reference substances 。

[0016] 1.4 Information of samples 。

[0017] Investigation on the construction method of the characteristic fingerprint of Jinzhen Oral Liquid in Example 1

[0018] 1.1 Investigation of test sample preparation methods 1.1.1 Investigation of test sample solvents Chromatographic conditions Chromatographic column: Topsil-C18 (4.6x250mm, 5μm); acetonitrile-methanol (70:35) as mobile phase A, 0.05% phosphoric acid solution (0.3g of sodium dodecyl sulfate added per 100ml) as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 230nm. The theoretical plate number calculated based on the baicalin peak should be no less than 5000; .

[0019] Preparation of reference solution Take appropriate amount of gallic acid, baicalin, wogonin, ammonium glycyrrhizinate 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.

[0020] Preparation of test solution Take 3 ml of Jinzhen oral solution (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 mixed solution for 15 minutes, and the ultrasonic parameters are power 200W and frequency 40kHz; centrifuge at 8000rpm 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 8000rpm 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 methanol and 5 ml water in turn; load the supernatant after centrifugation into the column at a flow rate of 1 ml / min, wash impurities with 5 ml water and 5 ml 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 to near dryness under reduced pressure at 40°C, and 1 ml of ethanol was added to dissolve the residue; 3 ml of deionized water precooled at 4°C was slowly added to the ethanol solution, and the mixture was allowed to stand overnight at 4°C, and centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected; the crystals were dissolved with 1 ml of methanol, vortexed and mixed, and then filtered through a 0.22 μm organic filter membrane, and the filtrate was taken as the test solution.

[0021] Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0022] like Figure 1As shown, the solvent investigation results show that when the extraction solvent contains 0.1% formic acid and the volume ratio of the extraction solvent is 70:30:0.1, the peak separation is better and the peak shape is better; when there is no formic acid in the extraction solvent, there are problems such as poor peak shape and peak missing, and the corresponding effects cannot be achieved. Therefore, the methanol-water-formic acid solution with a volume ratio of 70:30:0.1 is selected as the extraction solvent.

[0023] Table 1 Results of relative retention time of solvent investigation 。

[0024] 1.2 Chromatographic condition investigation 1.2.1 Wavelength investigation Chromatographic conditions Chromatographic column: Topsil-C18 (4.6x250mm, 5um); acetonitrile-methanol (70:35) as mobile phase A, 0.05% phosphoric acid solution (adding 0.3g sodium dodecyl sulfate per 100ml) as mobile phase B, and gradient elution is carried out according to the regulations in the following table; column temperature is 30°C; detection wavelengths are 210nm and 230nm. The number of theoretical plates calculated by the baicalin peak should be not less than 5000; 。

[0025] Preparation of reference substance solution Take appropriate amounts of gallic acid, baicalin, wogonin, ammonium glycyrrhizinate, and rhein reference substances, weigh accurately, and dissolve them in methanol to prepare solutions containing 75ug per 1ml respectively. Shake well to obtain the reference substance solution.

[0026] Preparation of test solution Take 3 ml of Jinzhen Oral Liquid, add 3 ml of a 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 stand for 10 minutes, centrifuge at 8000 rpm for 10 minutes, discard the precipitate, and retain the supernatant; purify the sample using a C18 solid-phase extraction column, and equilibrate the column with 5 ml of methanol and 5 ml of water in sequence; load the centrifuged supernatant onto the column at a flow rate of 1 ml / min, wash the impurities with 5 ml of water and 5 ml of 20% methanol in sequence, 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, add 1 ml of ethanol to dissolve the residue; slowly add 3 ml of deionized water pre-cooled to 4 °C to the ethanol solution, let stand overnight at 4 °C, centrifuge at 10000 rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with 1 ml of methanol, vortex mix and filter through a 0.22 μm organic filter membrane, and take the subsequent filtrate as the test solution.

[0027] Determination method Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0028] The results show that 11 characteristic peaks can be detected at wavelengths from 210 nm to 230 nm, and the RSD of the relative retention time is less than 1.0%. Therefore, the characteristic spectrum detection requirements can be met at wavelengths from 210 nm to 230 nm. The results are shown in the appendix Figure 2 。

[0029] Table 2 Results of relative retention time for wavelength investigation 。

[0030] 1.2.2 Investigation of mobile phase types Chromatographic condition 1: Chromatographic column: Topsil-C18 (4.6 x 250 mm, 5 μm); use acetonitrile-methanol (70:35) as mobile phase A and 0.05% phosphoric acid solution (adding 0.3 g of sodium dodecyl sulfate to every 100 ml) as mobile phase B, and perform gradient elution according to the regulations in the following table; column temperature is 30 °C; detection wavelength is 230 nm. The number of theoretical plates calculated based on the baicalin peak should be not less than 5000.

[0031] 。

[0032] Chromatographic conditions 2: Chromatographic column: Topsil-C18 (4.6x250mm, 5um); acetonitrile as mobile phase A, 0.05% phosphoric acid solution (adding 0.3g sodium dodecyl sulfate per 100ml) as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelength was 230nm. The theoretical plate number calculated by baicalin peak should be not less than 5000.

[0033] 。

[0034] Chromatographic conditions 3: Chromatographic column: Topsil-C18 (4.6x250mm, 5um); acetonitrile-methanol (70:35) as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelength was 230nm. The theoretical plate number calculated by baicalin peak should be not less than 5000.

[0035] 。

[0036] Preparation of reference substance solution Take appropriate amounts of gallic acid, baicalin, wogonin, ammonium glycyrrhizinate, and rhein reference substances, accurately weigh them, and separately dissolve them in methanol to prepare solutions containing 75ug per 1ml, shake well, and use them as reference substance solutions.

[0037] Preparation of test solution Take 3ml of Jinzhen Oral Liquid, add 3ml of methanol-water-formic acid composite solvent with a volume ratio of 70:30:0.1, vortex mix for 1 minute; ultrasonically treat the mixture for 15 minutes, ultrasonic parameters: power 200W, frequency 40kHz; centrifuge at 8000rpm for 10 minutes, take the supernatant; add 0.1mol / L ammonia water dropwise to the supernatant to adjust the pH to 7.0, let stand for 10 minutes, centrifuge at 8000rpm for 10 minutes, discard the precipitate, and retain the supernatant; purify the sample using a C18 solid-phase extraction column, balance the column with 5ml of methanol and 5ml of water in sequence; load the centrifuged supernatant onto the column at a flow rate of 1ml / min, wash the impurities with 5ml of water and 5ml of 20% methanol in sequence, discard the eluate; elute the target component with 5ml of 80% methanol containing 0.1% formic acid, collect the eluate; concentrate the eluate under reduced pressure to nearly dry at 40°C, add 1ml of ethanol to dissolve the residue; slowly add 3ml of deionized water pre-cooled at 4°C to the ethanol solution, let stand overnight at 4°C, centrifuge at 10000rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with 1ml of methanol, vortex mix and filter through a 0.22μm organic filter membrane, and take the subsequent filtrate as the test solution.

[0038] Determination method Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0039] The results showed that when acetonitrile - methanol (70:35) was used as mobile phase A and 0.05% phosphoric acid solution (containing 0.3 g of sodium dodecyl sulfate per 100 ml) was used as mobile phase B for elution, 11 characteristic peaks were presented in the chromatogram of the test sample, and the peak resolution was good and the peak shape was excellent; when acetonitrile was used as mobile phase A and 0.05% phosphoric acid solution (containing 0.3 g of sodium dodecyl sulfate per 100 ml) was used as mobile phase B or when acetonitrile - methanol (70:35) was used as mobile phase A and 0.05% phosphoric acid solution was used as mobile phase B for elution, each peak was missing and the corresponding effects could not be achieved.

[0040] Table 3 Results of the relative retention time for the investigation of the types of mobile phases 。

[0041] 1.2.3 Investigation of the mobile phase ratio Chromatographic conditions Chromatographic column: Topsil C18 (4.6 x 250 mm, 5 μm); acetonitrile - methanol (70:35) was used as mobile phase A and 0.05% phosphoric acid solution (containing 0.3 g of sodium dodecyl sulfate per 100 ml) was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the column temperature was 30 °C; the detection wavelength was 230 nm. The number of theoretical plates calculated based on the baicalin peak should be not less than 5000.

[0042] Mobile phase ratio 1: 。

[0043] Mobile phase ratio 2: 。

[0044] Mobile phase ratio 3: 。

[0045] Preparation of the reference solution Take appropriate amounts of gallic acid, baicalin, wogonin, ammonium glycyrrhizinate, and rhein reference substances, accurately weigh them, and dissolve them in methanol to prepare solutions containing 75 μg per 1 ml respectively. Shake well to obtain the reference solution.

[0046] Preparation of the test solution Take 3 ml of Jinzhen Oral Liquid, add 3 ml of a 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 dropwise 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; purify the sample using a C18 solid - phase extraction column, and equilibrate the column with 5 ml of methanol and 5 ml of water in sequence; load the centrifuged supernatant onto the column at a flow rate of 1 ml / min, wash the impurities with 5 ml of water and 5 ml of 20% methanol in sequence, 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, add 1 ml of ethanol to dissolve the residue; slowly add 3 ml of deionized water pre - cooled to 4 °C to the ethanol solution, let it stand overnight at 4 °C, centrifuge at 10000 rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with 1 ml of methanol, vortex - mix and filter through a 0.22 - μm organic filter membrane, and take the subsequent filtrate as the test solution.

[0047] Assay method Precisely pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0048] The results showed that when eluting with mobile - phase ratio 1, 11 characteristic peaks appeared in the chromatogram of the test solution, and the peak resolution was good and the peak shape was excellent; when eluting with mobile - phase ratio 2 or mobile - phase ratio 3, some peaks were missing and the corresponding effects could not be achieved.

[0049] Table 4 Results of relative retention time for investigation of mobile - phase ratio .

[0050] Example 2 Methodology verification of the characteristic fingerprint of Jinzhen Oral Liquid 2.1 Specificity Chromatographic conditions Chromatographic column: Topsil - C18 (4.6 x 250 mm, 5 μm); use acetonitrile - methanol (70:35) as mobile - phase A and 0.05% phosphoric acid solution (adding 0.3 g of sodium dodecyl sulfate per 100 ml) as mobile - phase B, and perform gradient elution according to the regulations in the following table; column temperature is 30 °C; detection wavelength is 230 nm. The number of theoretical plates calculated based on the baicalin peak should be not less than 5000.

[0051] .

[0052] Preparation of the reference solution Weigh appropriate amounts of gallic acid, baicalin, wogonin, ammonium glycyrrhizinate, and rhein reference substances accurately, and dissolve them in methanol respectively to prepare solutions containing 75 μg per 1 ml. Shake well to obtain the reference substance solution.

[0053] Preparation of Test Solution Take 3 ml of Jinzhen Oral Liquid, add 3 ml of a 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 a power of 200 W and a frequency of 40 kHz; centrifuge at 8000 rpm for 10 minutes and take the supernatant; add 0.1 mol / L ammonia water dropwise 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 a C18 solid-phase extraction column to purify the sample, and equilibrate the column with 5 ml of methanol and 5 ml of water in sequence; load the centrifuged supernatant onto the column at a flow rate of 1 ml / min, wash the impurities with 5 ml of water and 5 ml of 20% methanol in sequence, and discard the effluent; elute the target components with 5 ml of 80% methanol containing 0.1% formic acid, collect the eluate; concentrate the eluate under reduced pressure to nearly dry at 40 °C, add 1 ml of ethanol to dissolve the residue; slowly add 3 ml of deionized water precooled at 4 °C to the ethanol solution, let it stand overnight at 4 °C, centrifuge at 10000 rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with 1 ml of methanol, vortex mix and filter through a 0.22 μm organic filter membrane, and take the subsequent filtrate as the test solution.

[0054] Preparation of Negative Solution Take appropriate amounts of excipients, add 3 ml of a 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 a power of 200 W and a frequency of 40 kHz; centrifuge at 8000 rpm for 10 minutes and take the supernatant; add 0.1 mol / L ammonia water dropwise 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 a C18 solid-phase extraction column to purify the sample, and equilibrate the column with 5 ml of methanol and 5 ml of water in sequence; load the centrifuged supernatant onto the column at a flow rate of 1 ml / min, wash the impurities with 5 ml of water and 5 ml of 20% methanol in sequence, and discard the effluent; elute the target components with 5 ml of 80% methanol containing 0.1% formic acid, collect the eluate; concentrate the eluate under reduced pressure to nearly dry at 40 °C, add 1 ml of ethanol to dissolve the residue; slowly add 3 ml of deionized water precooled at 4 °C to the ethanol solution, let it stand overnight at 4 °C, centrifuge at 10000 rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with 1 ml of methanol, vortex mix and filter through a 0.22 μm organic filter membrane, and take the subsequent filtrate as the test solution.

[0055] Determination Method Precisely pipette 10 μl each of the negative solution, reference solution, and test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0056] The results showed that the negative solution had no interference and the specificity was good. The results are shown in the appendix Figure 3 .

[0057] 2.2 Precision Take 3 ml of Jinzhen Oral Liquid (batch number: 230257), prepare the test solution according to the preparation method of the test solution under item "2.1", inject 6 needles according to the chromatographic conditions under item "2.1", record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0058] Table 5 Results of relative retention time for precision .

[0059] Table 6 Results of relative peak area for precision .

[0060] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 10.0%, indicating good precision. The chromatogram is shown in the appendix Figure 4 .

[0061] 2.3 Repeatability Take 3 ml of Jinzhen Oral Liquid (batch number: 230257), a total of 6 portions, prepare the test solution according to the preparation method of the test solution under item "2.1", inject according to the chromatographic conditions under item "2.1", record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0062] Table 7 Results of relative retention time for repeatability .

[0063] Table 8 Results of relative peak area for repeatability .

[0064] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 10.0%, indicating good repeatability. The chromatogram is shown in the appendix Figure 5 .

[0065] 2.4 Stability Take 3 ml of Jinzhen Oral Liquid (batch number: 230257), prepare the test solution according to the preparation method of the test solution under item "2.1", inject according to the chromatographic conditions under item "2.1" at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively, record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0066] Table 9 Results of relative retention time for stability 。

[0067] Table 10 Results of relative peak area for stability 。

[0068] The results showed that the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 10.0%, indicating good solution stability. The chromatogram is shown in the appendix Figure 6 。

[0069] In summary, the specificity of this characteristic chromatogram is good, and the negative sample has no interference, indicating that the characteristic detection method has strong specificity; in the precision test, the RSD of the relative retention time is less than 1.0%, and the RSD of the relative peak area is less than 10.0%, indicating good instrument precision; in the repeatability test, the RSD of the relative retention time of each chromatographic peak is less than 1.0%, and the RSD of the relative peak area is less than 10.0%, indicating good repeatability; in the stability test, the RSD of the relative retention time of each chromatographic peak is less than 1.0%, and the RSD of the relative peak area is less than 10.0%. Therefore, the test solution is stable within 24 hours. This method has been well verified by methodology

[0070] Construction of the control characteristic chromatogram of Jinzhen Oral Liquid in Example 3 Chromatographic conditions Chromatographic column: Topsil-C18 (4.6x250mm, 5um); acetonitrile-methanol (70:35) was used as mobile phase A, and 0.05% phosphoric acid solution (0.3g of sodium dodecyl sulfate was added to every 100ml) was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the column temperature was 30°C; the detection wavelength was 230nm. The number of theoretical plates calculated by the baicalin peak should be not less than 5000

[0071] 。

[0072] Preparation of the reference solution Appropriate amounts of gallic acid, baicalin, wogonin, ammonium glycyrrhizinate, and rhein reference substances were accurately weighed and dissolved in methanol to prepare solutions containing 75ug per 1ml respectively, and shaken well to obtain the reference solution

[0073] Preparation of the test solution Take 3 ml of Jinzhen Oral Liquid, add 3 ml of a 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 drop - by - drop 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; purify the sample using a C18 solid - phase extraction column, and equilibrate the column with 5 ml of methanol and 5 ml of water in sequence; load the centrifuged supernatant onto the column at a flow rate of 1 ml / min, wash the impurities with 5 ml of water and 5 ml of 20% methanol in sequence, 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 until nearly dry, add 1 ml of ethanol to dissolve the residue; slowly add 3 ml of deionized water precooled at 4 °C to the ethanol solution, let it stand overnight at 4 °C, centrifuge at 10000 rpm for 15 minutes, discard the supernatant, and collect the crystals; dissolve the crystals with 1 ml of methanol, vortex - mix and then filter through a 0.22 - μm organic filter membrane, and take the subsequent filtrate as the test solution.

[0074] Determination method Precisely pipette 10 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0075] Import the chromatograms of the test samples of 8 batches of Jinzhen Oral Liquid into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", select the chromatographic peaks that exist in the chromatograms of different batches of Jinzhen Oral Liquid as the common peaks; generate the reference characteristic fingerprint using the average value calculation method, and calculate the relative retention time and relative peak area of each common peak. The results are shown in the appendix Figures 7 - 8 。

[0076] Table 11 Results of relative retention time of samples of eight batches of Jinzhen Oral Liquid 。

[0077] Table 12 Results of relative peak area of samples of eight batches of Jinzhen Oral Liquid 。

[0078] The chromatograms of the test samples of 8 batches of Jinzhen Oral Liquid all show 11 common peaks. The RSD of the relative retention time of the 11 common peaks is less than 1.0%, and the RSD of the relative peak area is less than 10.0%.

[0079] A total of 11 characteristic peaks were presented in the chromatogram of the test sample. Among them, peak 3, peak 8, peak 9, peak 10, and peak 11 corresponded to the reference peaks of gallic acid, baicalin, wogonin, ammonium glycyrrhizinate, and rhein, respectively; the peak corresponding to the reference peak of baicalin was the S peak. Calculate the relative retention time of each characteristic peak with respect to the S peak, and its relative retention time should be within the range of ±10% of the specified value. 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), 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: Take the reference substance, weigh it accurately, add methanol to make a solution containing 75ug per 1ml, shake well, and use it as the reference solution; the reference substances are: gallic acid, baicalin, wogonin, ammonium glycyrrhizinate, and rhein; (2) Preparation of test solution: Take Jinzhen oral liquid, 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 with 5 ml water and 5 ml The impurities were washed with 20% methanol and the effluent was discarded; the target component was eluted with 5 ml of 80% methanol containing 0.1% formic acid and the eluate was collected; the eluate was concentrated to near dryness under reduced pressure at 40°C, and then ethanol was added to dissolve the residue; deionized water precooled at 4°C was slowly added to the mixed solution of ethanol and the residue, and the mixture was allowed to stand at 4°C overnight, centrifuged at 10000 rpm for 15 minutes, the supernatant was discarded, and the crystals were collected; the crystals were dissolved with methanol, vortexed and mixed, and then filtered through a 0.22 μm organic filter membrane, and the filtrate was taken 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, 10ul of each sample was 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, 0.05% phosphoric acid solution with 0.3g of sodium dodecyl sulfate added per 100ml as the mobile phase B, and elution was performed according to the specified gradient; the column temperature was 30°C; the flow rate was 1ml per minute; the UV-visible detector had a detection wavelength of 210-230nm; The elution gradient conditions were: 0-8min, mobile phase A:B, volume ratio changed from 5:95 to 11:89; 8-9 min, the volume ratio of mobile phase A:phase B changed from 11:89 to 20:80; 9-23min, 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; 37-45min, the volume ratio of mobile phase A:phase B changed from 37:63 to 45:55; 45-60 min, the volume ratio of mobile phase A:B changed from 45:55 to 100:0; 60-63min, the volume ratio of mobile phase A:B 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 is 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 reference 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 glycyrrhizinate, and peak 11 corresponding to rhein; wherein the relative retention times of the 11 chromatographic peaks are respectively specified 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.

Images