Establishment method and application of children cough and asthma oral liquid fingerprint spectrum

Abstract

The invention provides an establishment method and application of a fingerprint spectrum of a children cough and asthma oral liquid, and belongs to the technical field of traditional Chinese medicine, the establishment method comprises the following steps: S1, dissolving the children cough and asthma oral liquid in an ethanol aqueous solution, extracting, filtering, taking filtrate, drying to obtain an extract, and dissolving the extract in a methanol aqueous solution to obtain a test solution; s2, respectively taking chlorogenic acid reference substances, cryptochlorogenic acid reference substances, liquiritin reference substances and ammonium glycyrrhizinate reference substances to prepare reference substance solutions; and S3, respectively and precisely sucking the reference solution and the test solution, performing sample introduction, performing determination by using a high performance liquid chromatograph, and establishing the fingerprint spectrum of the infantile cough and asthma oral liquid. The method is good in separation degree, accurate, reliable, simple to operate, high in precision, good in specificity, stability and repeatability, the established fingerprint spectrum of the children cough and asthma oral liquid is good in stability, and the quality of the children cough and asthma oral liquid can be more perfectly and scientifically controlled.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, relates to Xiao'er Kechuanling Oral Liquid, and particularly relates to a method for establishing a fingerprint of Xiao'er Kechuanling Oral Liquid and its application. Background Technique

[0002] Xiao'er Kechuanling Oral Liquid is a traditional Chinese medicine preparation, mainly used for treating cough symptoms in children caused by upper respiratory tract infections. The main raw materials are Ephedrae Herba, Lonicerae Japonicae Flos, Armeniacae Semen Amarum, Isatidis Radix, Gypsum Fibrosum, Glycyrrhizae Radix Et Rhizoma, and Trichosanthis Fructus, and the excipients are Stevioside and Sodium Benzoate. This medicine has the effects of dispersing the lung qi, clearing heat, relieving cough, and resolving phlegm. By clearing heat and detoxifying, dispersing the lung qi and relieving asthma, resolving phlegm and relieving cough, anti-inflammatory and antiviral, and improving respiratory symptoms, it can effectively relieve symptoms such as cough and asthma in children.

[0003] In Xiao'er Kechuanling Oral Liquid, Glycyrrhizae Radix Et Rhizoma is one of the components. Its active ingredient ammonium glycyrrhizinate has the effects of anti-inflammatory, immunomodulatory, antiviral, antitussive and expectorant, and protecting the membrane structure, and can relieve the cough symptoms caused by upper respiratory tract infections. The active ingredients of Glycyrrhizae Radix Et Rhizoma also include liquiritin, which has antioxidant, anti-inflammatory, and antibacterial effects and can relieve the cough symptoms caused by upper respiratory tract infections.

[0004] In Xiao'er Kechuanling Oral Liquid, Lonicerae Japonicae Flos is one of the components. Its active ingredient chlorogenic acid has the effects of antioxidant, anti-inflammatory, antiviral, antibacterial, clearing heat and detoxifying, and improving respiratory symptoms, and can relieve the cough symptoms caused by upper respiratory tract infections. The active ingredients of Lonicerae Japonicae Flos also include cryptochlorogenic acid, which can play anti-inflammatory and anti-inflammatory effects by inhibiting the activity of hyaluronidase (HAase), and also has antibacterial and antiviral, antioxidant, clearing heat and detoxifying, improving peptide hormone secretion defects, and restoring the normal physiological functions of cells.

[0005] The components of Xiao'er Kechuanling Oral Liquid are complex. When simultaneously detecting chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate as important active ingredients, other co-existing components will interfere with the detection results. Therefore, if a method can be established to simultaneously detect chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate in Xiao'er Kechuanling Oral Liquid and effectively shield the interference of other co-existing components, it will play an important role in promoting the quality control of Xiao'er Kechuanling Oral Liquid.

[0006] In the prior art, high performance liquid chromatography is mostly used to detect the active ingredients in Xiao'er Kechuanling Oral Liquid. For example:

[0007] The journal literature (Zhang Ting, Simultaneous determination of the contents of chlorogenic acid and liquiritin in Xiao'er Kechuanling Oral Liquid by high performance liquid chromatography, 2016) disclosed a method for simultaneously determining the contents of chlorogenic acid and liquiritin in Xiao'er Kechuanling Oral Liquid, using Agi-lent Eclipse Plus C 18A chromatographic column (150 mm × 4.6 mm, 5 μm). Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was carried out under the following conditions: 0 - 10 min, mobile phase A 10%, mobile phase B 90%; 10 - 15 min, mobile phase A 10% - 17%, mobile phase B 90% - 83%; 15 - 44 min, mobile phase A 17%, mobile phase B 83%; 44 - 45 min, mobile phase A 17% - 10%, mobile phase B 83% - 90%; 45 - 64 min, mobile phase A 10%, mobile phase B 90%. The flow rate was 1.0 mL·min -1 . Wavelength switching detection was performed, with the detection wavelengths being 327 nm (0 - 16 min, for determining chlorogenic acid) and 217 nm (16 - 64 min, for determining liquiritin). The injection volume was 10 μL. The column temperature was 30 °C. This method only detected the contents of chlorogenic acid and liquiritin and could not accurately detect many other chemical components. At the same time, during the detection process, the wavelengths were changed to detect chlorogenic acid and liquiritin separately. However, during the wavelength change process, the detector needed a certain amount of time to stabilize and achieve the purpose of accurate detection, making the detection process cumbersome.

[0008] The invention patent with the publication number CN107894488A discloses a quality control method for Xiao'er Kechuanling Oral Liquid, including the identification of Ephedrae Herba, Flos Lonicerae, Semen Armeniacae Amarum, Radix Isatidis, Glycyrrhizae Radix et Rhizoma, and Fructus Trichosanthis, as well as the detection of the contents of ephedrine hydrochloride and pseudoephedrine hydrochloride. Among them, the identification of Ephedrae Herba, Flos Lonicerae, Semen Armeniacae Amarum, Glycyrrhizae Radix et Rhizoma, and Fructus Trichosanthis adopted thin-layer chromatography. The identification of Radix Isatidis adopted high-performance liquid chromatography, and the chromatographic conditions were as follows: using octadecylsilane-bonded silica gel as the filler, the detection wavelength was 245 nm, and the flow rate was 1 mL / min; gradient elution was carried out with methanol and water according to the following conditions: 0 - 3 min, methanol 3% - water 97%; 3 - 20 min, methanol 10% - water 90%; 20 - 40 min, methanol 70% - water 30%; 40 - 50 min, methanol 70% - water 30%; ultraviolet detector, the detection wavelength was 245 nm. Among them, the detection of the contents of ephedrine hydrochloride and pseudoephedrine hydrochloride adopted high-performance liquid chromatography, and the chromatographic conditions were as follows: using polar ether-linked phenyl-bonded silica gel as the filler; using an acetonitrile - 0.1% triethylamine and 0.2% phosphoric acid aqueous solution with a volume ratio of 3:97 as the mobile phase; ultraviolet detector, the detection wavelength was 210 nm. This method did not involve the detection methods for chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate. At the same time, this method used different conditions to detect the contents of different components, making the detection steps cumbersome.

[0009] In summary, the prior art has not disclosed the detection methods for chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate in Xiao'er Kechuanling Oral Liquid.

[0010] Furthermore, the main ingredients of Xiao'er Kechuanling Granules are Ephedrae Herba, Lonicerae Japonicae Flos, Armeniacae Semen Amarum, Isatidis Radix, Gypsum Fibrosum, Glycyrrhizae Radix et Rhizoma, and Trichosanthis Fructus, which are the same as those of Xiao'er Kechuanling Oral Liquid. Both have the effects of dispersing lung qi, clearing heat, relieving cough, and resolving phlegm, and are used for cough caused by upper respiratory tract infection. Multiple detection methods for the active ingredients in Xiao'er Kechuanling Granules have been disclosed in the prior art.

[0011] For example: The UPLC fingerprint of Xiao'er Kechuanling Granules established in the journal literature (Lu Linling, Quality Evaluation of Xiao'er Kechuanling Granules Based on UPLC Fingerprint Combined with Chemometrics, 2022) has 18 common peaks, and 8 components ((R,S)-goitrin, chlorogenic acid, cryptochlorogenic acid, luteoloside, liquiritin, isochlorogenic acid A, isochlorogenic acid C, glycyrrhizic acid) were identified. The chromatographic conditions were as follows: Chromatographic column: Waters Acquity UPLC HSS T3 (2.1×100mm, 1.8μm); Mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution: 0 - 4.5min, 5% - 6% A; 4.5 - 12.0min, 6% - 20% A; 12.0 - 17.0min, 20% - 30% A; 17.0 - 21.0min, 30% - 45% A; 21.0 - 22.0min, 45% - 90% A; 22.0 - 24.0min, 90% A; 24.0 - 24.1min, 90% - 5% A; 24.1 - 26.0min, 5% A; Flow rate: 0.3mL·min -1 ; Column temperature: 35°C; Detection wavelength: 245nm; Injection volume: 1μL. However, the content of ammonium glycyrrhizinate was not detected simultaneously by this method.

[0012] The characteristic chromatogram of Xiao'er Kechuanling Granules established in the journal literature (Liang Youdan, Exploration of the potential mechanism of action of Xiao'er Kechuanling Granules in the treatment of cough based on characteristic chromatogram-network pharmacology-experimental verification, 2023) identified 21 common peaks in total. Seven chromatographic peaks, namely ephedrine hydrochloride, chlorogenic acid, amygdalin, liquiritin, luteoloside, 4,5-di-O-caffeoylquinic acid, and ammonium glycyrrhizinate, were identified by reference substances. Chromatographic conditions: YMC-Pack ODS-A-C18 chromatographic column (250 mm × 4.6 mm, 5 μm); acetonitrile (A)-0.02 mol / L potassium dihydrogen phosphate (0.1% phosphoric acid + 0.1% triethylamine) (B) as the mobile phase, gradient elution, flow rate 1.0 mL / min; wavelengths were 210 nm and 237 nm; column temperature was 30 °C; injection volume was 10 μL; elution time was 103 min, and the elution program was as follows: 0 min - 15 min, 2% A; 15 min - 50 min, 2% - 8% A; 50 min - 65 min, 8% - 11% A; 65 min - 68 min, 11% - 16% A; 68 min - 79 min, 16% - 23% A; 79 min - 80 min, 23% - 24% A; 80 min - 85 min, 24% - 25% A; 85 min - 98 min, 25% - 45% A; 98 min - 103 min, 45% - 2% A. However, the content of cryptochlorogenic acid was not detected simultaneously in this method, and the double-wavelength detection method was used to detect Xiao'er Kechuanling Granules. Affected by wavelength selection and interfering components, the equipment was complex and there were certain limitations.

[0013] The above technologies disclose various detection methods for the active ingredients in Xiao'er Kechuanling Granules. However, there are certain differences between Xiao'er Kechuanling Oral Liquid and Xiao'er Kechuanling Granules in steps such as extraction, concentration, paste preparation, and forming.

[0014] During the extraction process, in the preparation of Xiao'er Kechuanling Oral Liquid: First, medicinal materials such as bitter almonds, gypsum, isatis root, licorice, and trichosanthes fruit are decocted in water for 1 hour, and the medicinal liquid is taken after filtration; then ephedra and honeysuckle are added to the above medicinal residues and decocted for another 1 hour, and the extraction liquids are combined. In the preparation of Xiao'er Kechuanling Granules: First, ephedra, bitter almonds, gypsum, and raw licorice are weighed and decocted in water for 1 hour, and then filtered; then the medicinal residues are decocted with isatis root, honeysuckle, and trichosanthes fruit in water, and the extraction liquids are combined.

[0015] During the concentration and paste preparation process, for Xiao'er Kechuanling Oral Liquid: The extraction liquid is filtered through a ceramic membrane, and the clear liquid is collected and concentrated to make an extract. For Xiao'er Kechuanling Granules: It usually involves concentrating the extraction liquid, mixing it with dextrin and powdered sugar, and preparing granules through a one-step granulation process.

[0016] During the forming process, for Xiao'er Kechuanling Oral Liquid: after preparing the extract, add flavoring agents (such as stevioside, sodium benzoate) and purified water to a certain volume, stir evenly, and then filter through a tubular ultrafiltration membrane to obtain the final oral liquid product; for Xiao'er Kechuanling Granules: after preparing the paste, prepare the granules through a one-step granulation process, including steps such as crushing, sieving, total mixing, and packaging, and control the moisture content of the granules.

[0017] Due to the above differences, there are certain differences in the active ingredients and interfering components between Xiao'er Kechuanling Oral Liquid and Xiao'er Kechuanling Granules. Therefore, there are also certain differences in the methods for establishing the fingerprint spectra of Xiao'er Kechuanling Oral Liquid and Xiao'er Kechuanling Granules. The prior art has not disclosed the detection methods for chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate in Xiao'er Kechuanling Oral Liquid. The components of Xiao'er Kechuanling Oral Liquid are complex. As important active ingredients, establishing a method that can simultaneously detect chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate in Xiao'er Kechuanling Oral Liquid and effectively shield the interference of other coexisting components has an important promoting effect on the quality control of Xiao'er Kechuanling Oral Liquid. Summary of the Invention

[0018] In view of the problems existing in the prior art, the present invention provides a method for establishing the fingerprint spectrum of Xiao'er Kechuanling Oral Liquid and its application. The present invention proposes a detection method for Xiao'er Kechuanling Oral Liquid with strong specificity and high accuracy.

[0019] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0020] In the first aspect, the present invention provides a method for establishing the fingerprint spectrum of Xiao'er Kechuanling Oral Liquid, including the following steps:

[0021] S1: Dissolve Xiao'er Kechuanling Oral Liquid in an ethanol aqueous solution for extraction, filter, take the filtrate, dry to obtain an extract, and dissolve the extract in a methanol aqueous solution to obtain a test solution;

[0022] S2: Respectively take reference substances of chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate to prepare reference solutions;

[0023] S3: Precisely absorb the reference solution and the test solution respectively, inject the samples, and use a high-performance liquid chromatograph to determine and establish the fingerprint spectrum of Xiao'er Kechuanling Oral Liquid.

[0024] In the step S1, the ethanol aqueous solution is an 80% ethanol aqueous solution; the methanol aqueous solution is a 50% methanol aqueous solution.

[0025] Preferably, the chromatographic conditions of the high-performance liquid chromatograph include:

[0026] Chromatographic column: packed with octadecylsilane chemically bonded silica gel;

[0027] Mobile phase A: acetonitrile;

[0028] Mobile phase B: 0.1% phosphoric acid solution or water;

[0029] Gradient elution;

[0030] Column temperature: 25 - 30 °C;

[0031] Flow rate: 0.5 - 1 mL / min;

[0032] Detection wavelength: 230 - 270 nm.

[0033] The conditions of the gradient elution are preferably:

[0034] 0 - 9 min, the volume fraction of mobile phase A is 5 - 6%, and the volume fraction of mobile phase B is 94 - 95%;

[0035] 9 - 16 min, the volume fraction of mobile phase A is 6 - 9%, and the volume fraction of mobile phase B is 91 - 94%;

[0036] 16 - 24 min, the volume fraction of mobile phase A is 9%, and the volume fraction of mobile phase B is 91%;

[0037] 24 - 35 min, the volume fraction of mobile phase A is 9 - 15%, and the volume fraction of mobile phase B is 85 - 91%;

[0038] 35 - 50 min, the volume fraction of mobile phase A is 15 - 20%, and the volume fraction of mobile phase B is 75 - 80%;

[0039] 50 - 70 min, the volume fraction of mobile phase A is 20 - 30%, and the volume fraction of mobile phase B is 70 - 80%;

[0040] 70 - 86 min, the volume fraction of mobile phase A is 30 - 45%, and the volume fraction of mobile phase B is 55 - 70%;

[0041] 86 - 90 min, the volume fraction of mobile phase A is 45%, and the volume fraction of mobile phase B is 55%;

[0042] 90 - 96 min, the volume fraction of mobile phase A is 45 - 90%, and the volume fraction of mobile phase B is 10 - 55%;

[0043] 96 - 100 min, the volume fraction of mobile phase A is 90 - 5%, and the volume fraction of mobile phase B is 10 - 95%;

[0044] 100 - 105 min, the volume fraction of mobile phase A is 5%, and the volume fraction of mobile phase B is 95%.

[0045] The chromatographic column is preferably a waters T3-C18 chromatographic column with a specification of 250 mm × 4.6 mm and 5 μm. The column temperature is preferably 30 °C. The flow rate is preferably 1 mL / min. The detection wavelength is preferably 245 nm.

[0046] The fingerprint of Xiao'er Kechuanling Oral Liquid consists of 14 fingerprint peaks in total. Among them, the 4th peak is identified as chlorogenic acid, the 7th peak is identified as cryptochlorogenic acid, the 10th peak is identified as liquiritin, and the 14th peak is identified as ammonium glycyrrhizinate.

[0047] In the second aspect, the present invention provides an application of the above-mentioned establishment method in the quality detection of Xiao'er Kechuanling Oral Liquid.

[0048] Compared with the prior art, the present invention has the following beneficial effects: The method for establishing the fingerprint of Xiao'er Kechuanling Oral Liquid of the present invention has good resolution, accuracy, reliability, simple operation, high precision, good specificity, stability and repeatability. The established fingerprint of Xiao'er Kechuanling Oral Liquid has good stability, and can more perfectly and scientifically control the quality of Xiao'er Kechuanling Oral Liquid. Description of the Drawings

[0049] Figure 1 It is the fingerprint of Example 1;

[0050] Figure 2 It is the fingerprint of Comparative Example 1;

[0051] Figure 3 It is the fingerprint of Comparative Example 2;

[0052] Figure 4 It is the fingerprint of Comparative Example 3;

[0053] Figure 5 It is the fingerprint of Comparative Example 4;

[0054] Figure 6 It is the fingerprint of Comparative Example 5;

[0055] Figure 7 It is the fingerprint of Xiao'er Kechuanling Oral Liquid;

[0056] Figure 8 It is the linear equation diagram of chlorogenic acid;

[0057] Figure 9 It is the linear equation diagram of ammonium glycyrrhizinate;

[0058] Figure 10 It is the fingerprint of samples with different batch numbers. Detailed Embodiments

[0059] It should be noted that, unless otherwise specified, the raw materials used in the present invention are all ordinary commercially available products, and their sources are not specifically limited.

[0060] Among them, the solvents are all chromatographic grade.

[0061] Example 1

[0062] Preparation of reference substance solution: Weigh appropriate amounts of chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate reference substances accurately, and prepare a solution containing 0.07 mg per 1 mL with 50% methanol to obtain the reference substance solution.

[0063] Preparation of test solution: Precisely measure 5 mL of Xiao'er Kechuanling Oral Liquid (batch number of the test sample: 215270012), accurately add 20 mL of 80% ethanol, extract ultrasonically for 30 min, with a power of 250 W and a frequency of 33 kHz, filter, take the filtrate, dry the filtrate to obtain the extract, with the drying temperature being 45 - 50 °C, dissolve the extract in 20 mL of 50% methanol, filter through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain the test solution.

[0064] Chromatographic conditions: Using octadecylsilane chemically bonded silica as the filler, adopt a waters T3 - C18 chromatographic column, with a specification of 250 mm × 4.6 mm, 5 μm; using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; perform gradient elution according to Table 1, with a column temperature of 30 °C, a flow rate of 1 mL / min, and a detection wavelength of 245 nm.

[0065] Table 1 Gradient elution conditions of Example 1

[0066]

[0067] The detection results are as Figure 1 shown. The retention time of chlorogenic acid is 31.793 min, the retention time of cryptochlorogenic acid is 35.039 min, the retention time of liquiritin is 51.085 min, and the retention time of ammonium glycyrrhizinate is 87.237 min. Figure 1 The results show that the separation effect of chlorogenic acid in Example 1 is good and meets the resolution requirements, as shown in Figure 1 .

[0068] Comparative Example 1

[0069] Preparation of reference substance solution: Weigh appropriate amounts of chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate reference substances accurately, and prepare a solution containing 0.07 mg per 1 mL with 50% methanol to obtain the reference substance solution.

[0070] Preparation of test solution: Accurately measure 5 mL of Xiao'er Kechuanling Oral Liquid (batch number of the test sample: 215270012), accurately add 20 mL of 80% ethanol, extract by ultrasound for 30 min with a power of 250 W and a frequency of 33 kHz, filter, take the filtrate, dry the filtrate to obtain an extract, with the drying temperature being 45 - 50 °C. Dissolve the extract in 20 mL of 50% methanol, filter through a 0.22 μm filter membrane, take the filtrate, and thus obtain the test solution.

[0071] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler, adopt a waters T3-C18 chromatographic column with a specification of 250 mm × 4.6 mm, 5 μm; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; perform gradient elution according to Table 2, with the column temperature being 30 °C, the flow rate: 1 mL / min, and the detection wavelength being 245 nm.

[0072] Table 2 Gradient elution conditions of Comparative Example 1

[0073]

[0074] The detection results are as Figure 2 shown. The retention time of chlorogenic acid is 19.308 min, the retention time of liquiritin is 60.248 min, and the retention time of ammonium glycyrrhizinate is 72.945 min. Figure 2 The results show that under the detection conditions of Comparative Example 1, the resolution of chlorogenic acid is not good and still needs to be further optimized.

[0075] Comparative Example 2

[0076] Preparation of reference solution: Accurately weigh appropriate amounts of reference substances of chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate respectively, add 50% methanol to make a solution containing 0.07 mg per 1 mL, and thus obtain the reference solution.

[0077] Preparation of test solution: Accurately measure 5 mL of Xiao'er Kechuanling Oral Liquid (batch number of the test sample: 215270012), accurately add 20 mL of 80% ethanol, extract by ultrasound for 30 min with a power of 250 W and a frequency of 33 kHz, filter, take the filtrate, dry the filtrate to obtain an extract, with the drying temperature being 45 - 50 °C. Dissolve the extract in 20 mL of 50% methanol, filter through a 0.22 μm filter membrane, take the filtrate, and thus obtain the test solution.

[0078] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler, adopt a Welch Materials AQ-C18 chromatographic column with a specification of 250 mm × 4.6 mm, 5 μm; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; perform gradient elution according to Table 3, with the column temperature being 25 °C, the flow rate: 1 mL / min, and the detection wavelength being 245 nm.

[0079] Table 3 Gradient elution conditions for Comparative Example 2

[0080]

[0081]

[0082] The detection results are as Figure 3 shown. The retention time of chlorogenic acid is 34.094 min, and the retention time of ammonium glycyrrhizinate is 95.351 min. Figure 3 The results show that chlorogenic acid in Comparative Example 2 is still not separated, and the resolution is insufficient.

[0083] Comparative Example 3

[0084] Preparation of reference substance solution: Weigh appropriate amounts of reference substances of chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate accurately, and prepare a solution containing 0.07 mg per 1 mL with 50% methanol to obtain the reference substance solution.

[0085] Preparation of test solution: Accurately measure 5 mL of Xiao'er Kechuanling Oral Liquid (batch number of the test sample: 215270012), accurately add 20 mL of 80% ethanol, extract ultrasonically for 30 min, with a power of 250 W and a frequency of 33 kHz, filter, take the filtrate, dry the filtrate to obtain the extract, with the drying temperature at 45 - 50 °C, dissolve the extract in 20 mL of 50% methanol, filter through a 0.22 μm filter membrane, take the filtrate to obtain the test solution.

[0086] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler, adopt a waters T3-C18 chromatographic column, with a specification of 250 mm × 4.6 mm, 5 μm; using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; perform gradient elution according to Table 4, with a column temperature of 25 °C, a flow rate of 1 mL / min, and a detection wavelength of 245 nm.

[0087] Table 4 Gradient elution conditions for Comparative Example 3

[0088]

[0089] The detection results are as Figure 4 shown. The retention time of chlorogenic acid is 29.954 min, the retention time of cryptochlorogenic acid is 33.884 min, the retention time of liquiritin is 48.694 min, and the retention time of ammonium glycyrrhizinate is 80.240 min. Figure 4 The results show that the resolution of chlorogenic acid in Comparative Example 3 is insufficient.

[0090] Comparative Example 4

[0091] Preparation of reference substance solution: Weigh appropriate amounts of chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate reference substances accurately, and prepare a solution containing 0.07 mg per 1 mL with 80% ethanol to obtain the reference substance solution.

[0092] Preparation of test solution: Precisely measure 5 mL of Xiao'er Kechuanling Oral Liquid (batch number of the test sample: 215270012), accurately add 20 mL of 80% ethanol, extract by ultrasonic for 30 min with a power of 250 W and a frequency of 33 kHz, filter, take the filtrate, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0093] Detection was carried out according to the chromatographic conditions of Example 1, and the results were as Figure 5 shown. The retention time of chlorogenic acid was 31.730 min, and the retention time of the ammonium glycyrrhizinate peak was 95.285 min. It was found that the resolution of chlorogenic acid in the chromatogram of Comparative Example 4 was insufficient.

[0094] Comparative Example 5

[0095] Preparation of reference substance solution: Weigh appropriate amounts of chlorogenic acid, cryptochlorogenic acid, liquiritin, and ammonium glycyrrhizinate reference substances accurately, and prepare a solution containing 0.07 mg per 1 mL with 50% methanol to obtain the reference substance solution.

[0096] Preparation of test solution: Precisely measure 5 mL of Xiao'er Kechuanling Oral Liquid (batch number of the test sample: 215270012), accurately add 20 mL of 50% methanol, extract by ultrasonic for 30 min with a power of 250 W and a frequency of 33 kHz, filter, take the filtrate, filter through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain the test solution.

[0097] Detection was carried out according to the chromatographic conditions of Example 1, and the results were as Figure 6 shown. The retention time of chlorogenic acid was 31.844 min, and the retention time of the ammonium glycyrrhizinate peak was 95.351 min. It was found that there were many impurity peaks and large baseline noise in the chromatogram of Comparative Example 5.

[0098] Test Example 1

[0099] Methodology verification

[0100] Methodology verification was carried out on the fingerprint and multi-index component detection methods according to the reference substance solution and test solution prepared in Example 1 and the determined chromatographic conditions, as follows:

[0101] 1. Verification of the methodology for fingerprint inspection:

[0102] 1.1 Investigation of precision

[0103] Six portions of Xiao'er Kechuanling Oral Liquid (batch number of the test sample: 215270012) were taken to prepare the test sample solution. Liquid chromatography detection was carried out, and injection was performed continuously for 6 times, named replicate 1 - 6 respectively. The injection volume was 10 μL, and the consistency of the retention time and chromatographic peak area of each main chromatographic peak was investigated. The results of the precision investigation are shown in Table 5.

[0104] The results showed that the RSD of the retention time was between 0.01 - 0.69%, and the RSD of the chromatographic peak area was between 0.2% - 3.53%, indicating good precision.

[0105] Table 5 Results of the precision investigation of the fingerprint

[0106]

[0107]

[0108] 1.2 Repeatability investigation

[0109] Six portions of Xiao'er Kechuanling Oral Liquid (batch number of the test sample: 215270012) were taken to prepare the test sample solutions respectively, named S1 - S6. Liquid chromatography detection was carried out, and the injection volume was 10 μL. The consistency of the retention time and chromatographic peak area of each main chromatographic peak was investigated. The results of the repeatability investigation are shown in Table 6.

[0110] The results showed that the RSD of the retention time was between 0.01 - 0.69%, and the RSD of the chromatographic peak area was between 0.24% - 3.38%, indicating good repeatability.

[0111] Table 6 Results of the repeatability investigation of the fingerprint

[0112]

[0113]

[0114] 1.3 Solution stability investigation

[0115] Precisely pipette the test sample solution (batch number of the test sample: 215270012), and perform injection detection at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h, 72 h after preparation at room temperature. The injection volume was 10 μL, record the peak area of the sample, investigate the stability of the sample solution, and calculate the relative standard deviation RSD (%). The results of the solution stability are shown in Table 7.

[0116] The results showed that the RSD of the retention time was between 0.03% - 0.91%, and the RSD of the chromatographic peak area was between 0.21% - 4.90%, indicating good solution stability.

[0117] Table 7 Results of the investigation on the stability of the fingerprint solution

[0118]

[0119]

[0120] 2. Establishment of the fingerprint and designation of the reference peak

[0121] The fingerprint of Xiao'er Kechuanling Oral Liquid is as shown in Figure 7 As shown. The results show that a total of 14 common peaks appear in the control fingerprint. Among them, 4 common peaks are identified by comparing with the reference substance solution. Among them, peak 4 is chlorogenic acid, peak 7 is cryptochlorogenic acid, peak 10 is liquiritin, and peak 14 is ammonium glycyrrhizinate. According to the selection requirements of the fingerprint reference peak, the chromatographic peak of chlorogenic acid at peak 4 is stable, with good resolution and a large peak area. Therefore, peak 4 is determined as the reference peak.

[0122] 3. Methodology verification of multi-index component determination

[0123] 3.1 Specificity analysis

[0124] Negative sample: First, weigh the remaining six herbs without licorice and honeysuckle according to the prescription amount. Then, prepare two negative samples without licorice and honeysuckle respectively according to the prescription. Then, prepare the negative sample solution according to the preparation method of the test solution in Example 4.

[0125] The results of the specificity experiment are shown in Table 8. The results show that the retention times of the chromatographic peaks of chlorogenic acid and ammonium glycyrrhizinate in the test solution are the same as those of the chromatographic peaks of chlorogenic acid and ammonium glycyrrhizinate in the reference substance solution respectively. In the chromatogram of the negative sample, no corresponding peaks appear at the positions of the chromatographic peaks of chlorogenic acid and ammonium glycyrrhizinate in the reference substance solution. This indicates that the negative sample has no interference, indicating that this method has good specificity.

[0126] Table 8 Results of the specificity test

[0127]

[0128] 3.2 Linearity analysis

[0129] Prepare the chlorogenic acid linear solution: Accurately weigh the chlorogenic acid reference standard and dissolve it in 50% methanol to obtain a chlorogenic acid linear solution with a concentration of 513.6642 μg / mL. Then, dilute it to obtain chlorogenic acid linear solutions with concentrations of 205.4657 μg / mL, 154.0993 μg / mL, 128.4161 μg / mL, and 25.6832 μg / mL respectively, and perform liquid chromatography analysis.

[0130] The results of the linearity test are shown in Table 9 and Figure 8 as shown. Figure 8It is the linear equation graph of chlorogenic acid. The results show that within the range of 25.6832 μg / mL - 513.6642 μg / mL of chlorogenic acid, the linear equation for the linear relationship between the chlorogenic acid concentration and the peak area is y = 16635x - 71294, with R = 0.9996, indicating good linearity of the method.

[0131] Table 9 Results of the linearity test of chlorogenic acid

[0132]

[0133] Prepare the linear solution of ammonium glycyrrhizinate: Accurately weigh the ammonium glycyrrhizinate reference standard and dissolve it in 50% methanol to obtain a linear solution of chlorogenic acid with a concentration of 480.8736 μg / mL. Then dilute it to obtain linear solutions of ammonium glycyrrhizinate with concentrations of 240.4368 μg / mL, 144.2621 μg / mL, 96.1747 μg / mL, and 24.0437 μg / mL respectively, and conduct liquid chromatography analysis.

[0134] The results of the linearity test are shown in Table 10 and Figure 9 as follows, Figure 9 It is the linear equation graph of ammonium glycyrrhizinate. The results show that within the range of 24.5414 μg / mL - 490.8277 μg / mL of ammonium glycyrrhizinate, the linear equation for the linear relationship between the ammonium glycyrrhizinate concentration and the peak area is y = 7964.1X - 7508.2, with R = 0.9999, indicating good linearity of the method.

[0135] Table 10 Results of the linearity test of ammonium glycyrrhizinate

[0136]

[0137] 3.3 Accuracy analysis

[0138] 3.3.1 Chlorogenic acid addition recovery test

[0139] Accurately weigh the test solution of the same batch with known content (batch number: 215270012). Take 6 portions of the test solution, each 3 mL. Accurately add 1540.99 μg of chlorogenic acid reference standard to each sample, mix well to obtain the test samples, conduct liquid chromatography analysis, inject 10 μL, and calculate the recovery rate, average recovery rate, and RSD by the external standard method. The results are shown in Table 11.

[0140] The results show that the average recovery rate of chlorogenic acid is 98.43%, within the range of 85% - 110%. The RSD is 1.37%, less than 2.0%, meeting the verification requirements, indicating good accuracy of this method.

[0141] Table 11 Chlorogenic acid addition recovery test

[0142]

[0143]

[0144] 3.3.2 Ammonium Glycyrrhizinate Addition and Recovery Test

[0145] Accurately weigh the test solution of the same batch with known content (batch number: 215270012). Take 6 portions of the test solution, each 3 mL. Accurately add 2355.61 μg of ammonium glycyrrhizinate standard to each sample, mix well to obtain the sample to be tested, perform liquid chromatography analysis, inject 10 μL, and calculate the recovery rate, average recovery rate, and RSD by the external standard method. The results are shown in Table 12.

[0146] The results show that the average recovery rate of ammonium glycyrrhizinate is 97.45%, within the range of 85% - 110%. The RSD is 0.78%, less than 2.0%, meeting the verification requirements, indicating that the accuracy of this method is good.

[0147] Table 12 Ammonium Glycyrrhizinate Addition and Recovery Test

[0148]

[0149] 3.4 Precision Investigation

[0150] 3.4.1 Intermediate Precision Investigation

[0151] The results of the intermediate precision tests for chlorogenic acid and ammonium glycyrrhizinate are shown in Tables 13 - 14 respectively.

[0152] Table 13 shows that six portions of chlorogenic acid test solutions were prepared by two experimenters at different times, and the RSD values were 0.18% and 0.26% respectively; the RSD% of the intermediate precision was 0.44%, less than 8%, meeting the requirements, indicating that the intermediate precision of the chlorogenic acid content of the method is good.

[0153] Table 14 shows that six portions of ammonium glycyrrhizinate test solutions were prepared by two experimenters at different times, and the RSD values of the test results were 0.29% and 0.34% respectively; the RSD% of the intermediate precision was 0.30%, less than 8%, meeting the requirements, indicating that the intermediate precision of the ammonium glycyrrhizinate content of the method is good.

[0154] Table 13 Results of Intermediate Precision Test for Chlorogenic Acid

[0155]

[0156]

[0157] Table 14 Results of Intermediate Precision Test for Ammonium Glycyrrhizinate

[0158]

[0159] 3.5 Repeatability Investigation

[0160] The repeatability investigation experiment of chlorogenic acid content is shown in Table 15. The results show that the RSD of the repeatability of chlorogenic acid content detection is 0.26%, which meets the requirements.

[0161] Table 15 Repeatability Investigation of Chlorogenic Acid Content

[0162]

[0163] The repeatability investigation experiment of ammonium glycyrrhizinate content is shown in Table 16. The results show that the RSD of the repeatability of chlorogenic acid content detection is 0.34%, which meets the requirements.

[0164] Table 16 Repeatability Investigation of Ammonium Glycyrrhizinate Content

[0165]

[0166]

[0167] 3.6 Solution Stability Investigation

[0168] The test solution was placed at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h, and 72h respectively, and the stabilities of chlorogenic acid and ammonium glycyrrhizinate were detected.

[0169] The results of the stability test of chlorogenic acid in the test solution are shown in Table 17. The results in Table 17 show that under the specified conditions, the RSD value of the peak area of chlorogenic acid obtained from the test solution within 72 hours is 0.21%, which meets the requirements. Therefore, the chlorogenic acid in the test solution is relatively stable.

[0170] Table 17 Results of the Stability Test of Chlorogenic Acid in the Test Solution

[0171]

[0172] The results of the stability test of ammonium glycyrrhizinate in the test solution are shown in Table 18. The results in Table 18 show that under the specified conditions, the RSD value of the peak area of ammonium glycyrrhizinate obtained from the test solution within 72 hours is 0.35%, which meets the requirements. Therefore, the ammonium glycyrrhizinate in the test solution is relatively stable.

[0173] Table 18 Results of the Stability Test of Ammonium Glycyrrhizinate in the Test Solution

[0174]

[0175]

[0176] Test Example 2

[0177] Sample: Children's Kechuanling oral liquid, batch numbers are 235150012 ordinary type, 235150022 ordinary type, 235150032 ordinary type, 235270012 concentrated type, 235270022 concentrated type, 235270032 concentrated type, 235290012 Pharmacopoeia concentrated type, 235290022 Pharmacopoeia concentrated type, 235290032 Pharmacopoeia concentrated type.

[0178] The samples of different batches were tested using the verified test method in Example 5 (the reference solution and the test solution prepared in Example 4 and the determined chromatographic conditions), with the reference solution as the control group. Figure 10 As shown. The contents of chlorogenic acid and ammonium glycyrrhizinate in samples of different batches were determined, and the similarity of finished products of different batches was calculated. The evaluation method was as follows: the measured chromatogram of the pediatric Kechuanling oral liquid was imported into the "Chinese Medicine Fingerprint Evaluation System (2012)" software of the National Pharmacopoeia Committee, and the chromatograms of three batches of the preparation of pediatric Kechuanling oral liquid (batch numbers: 235150012, 235150022, 235150032) were set to generate a reference spectrum R under the software, and the chromatograms of the same process before change (batch numbers: 235150012, 235150022, 235150032, 235290012, 235290022, 235290032) and after change (batch numbers: 235270012, 235270022, 235270032) were matched to generate a similarity report for evaluation.

[0179] The similarity comparison results of samples of different batches and the contents of chlorogenic acid and ammonium glycyrrhizinate in the samples are shown in Tables 19 and 20, respectively. The results in Table 19 show that the similarity of samples with different batches is 0.995-1.000, not less than 0.90, and the similarity is high, indicating that the detection method of Example 1 has good stability. The results in Table 20 show that in samples with different batches, the content of chlorogenic acid is in the range of 0.45-0.91 mg / mL, and the content of ammonium glycyrrhizinate is in the range of 0.69-1.14 mg / mL.

[0180] Table 19 Similarity comparison results of samples from different batches

[0181]

[0182]

[0183] Table 20 Contents of chlorogenic acid and ammonium glycyrrhizinate in samples of different batches

[0184] Sample Name Chlorogenic Acid (mg / mL) Ammonium Glycyrrhizinate (mg / mL) Batch 23515001, Regular Type 0.45 0.69 Batch 23515002, Regular Type 0.45 0.69 Batch 23515003, Regular Type 0.45 0.69 Batch 23527001, Concentrated Type 0.91 1.39 Batch 23527002, Concentrated Type 0.91 1.41 Batch 23527003, Concentrated Type 0.91 1.40 Batch 23529001, Pharmacopoeia Concentrated Type 0.91 1.38 Batch 23529002, Pharmacopoeia Concentrated Type 0.90 1.37 Batch 23529003, Pharmacopoeia Concentrated Type 0.91 1.37

[0185] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of pediatric Kechuanling oral liquid, characterized in that: The following steps are involved: S1: Dissolve the Xiaoer Kechuanling oral liquid in an ethanol aqueous solution, extract, filter, take the filtrate, dry to obtain an extract, dissolve the extract in a methanol aqueous solution to obtain a test solution; S2: prepare reference solution by taking chlorogenic acid, cryptochlorogenic acid, liquiritin and ammonium glycyrrhizinate reference substances respectively; S3: Accurately aspirate the reference solution and the test solution respectively, inject the samples, and determine them using a high performance liquid chromatography to establish the fingerprint of the Children's Kechuanling oral liquid.

2. The method according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatograph include: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase A: acetonitrile; Mobile phase B: 0.1% phosphoric acid solution or water; Gradient elution; Column temperature 25-30°C; Flow rate: 0.5~1mL / min; Detection wavelength: 230~270nm.

3. The method according to claim 2, characterized in that: The conditions of the gradient elution are: 0-9min, the volume fraction of mobile phase A is 5-6%, and the volume fraction of mobile phase B is 94-95%; 9-16min, the volume fraction of mobile phase A is 6-9%, and the volume fraction of mobile phase B is 91-94%; 16-24min, the volume fraction of mobile phase A was 9%, and the volume fraction of mobile phase B was 91%; 24-35min, the volume fraction of mobile phase A is 9-15%, and the volume fraction of mobile phase B is 85-91%; 35-50min, the volume fraction of mobile phase A is 15-20%, and the volume fraction of mobile phase B is 75-80%; 50-70min, the volume fraction of mobile phase A is 20-30%, and the volume fraction of mobile phase B is 70-80%; 70-86min, the volume fraction of mobile phase A is 30-45%, and the volume fraction of mobile phase B is 55-70%; 86-90min, the volume fraction of mobile phase A was 45%, and the volume fraction of mobile phase B was 55%; 90-96min, the volume fraction of mobile phase A is 45-90%, and the volume fraction of mobile phase B is 10-55%; 96-100min, the volume fraction of mobile phase A is 90-5%, and the volume fraction of mobile phase B is 10-95%; 100-105min, the volume fraction of mobile phase A is 5%, and the volume fraction of mobile phase B is 95%.

4. The method according to claim 2, characterized in that: The chromatographic column is a Waters T3-C18 chromatographic column with specifications of 250 mm×4.6 mm and 5 μm.

5. The method according to claim 2, characterized in that: The column temperature was 30°C.

6. The method according to claim 2, characterized in that: The flow rate was 1 mL / min.

7. The method according to claim 2, characterized in that: The detection wavelength is 245 nm.

8. The method according to claim 1, characterized in that: The fingerprint spectrum of the pediatric Kechuanling oral liquid is composed of 14 fingerprint peaks, among which peak No. 4 is chlorogenic acid, peak No. 7 is cryptochlorogenic acid, peak No. 10 is glycyrrhizin, and peak No. 14 is ammonium glycyrrhizinate.

9. The method according to claim 1, characterized in that: In step S1, the ethanol aqueous solution is an 80% ethanol aqueous solution; the methanol aqueous solution is a 50% methanol aqueous solution.

10. Application of the method established in any one of claims 1 to 9 in the quality inspection of Kechuanling oral liquid for children.

Images