Method for detecting contents of multiple index components of coughing oral liquid

Abstract

The invention provides a method for detecting the contents of multiple index components of a coughing oral liquid, which comprises the following steps of: determining the contents of nine key pharmacodynamic index components, namely ephedrine hydrochloride, pseudoephedrine hydrochloride, apiose liquiritin, isoliquiritin, baicalin, huperzin A glycoside, wogonin, baicalein and wogonin; the detection method has the advantages of simplicity and convenience in operation, high stability, high pertinence and high practicability, can be used as a detection and evaluation method for production and internal quality components of the coughing oral liquid, and has a good market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of quality testing of chemical components of traditional Chinese medicine, and in particular relates to a method for detecting the content of multiple indicators of components in cough syrup oral liquid. Background Technology

[0002] Cough Syrup is a major compound traditional Chinese medicine preparation produced by Shaanxi Buchang Pharmaceutical Co., Ltd. It is made from eight traditional Chinese medicines, including Scutellaria baicalensis, Fritillaria cirrhosa, Eriobotrya japonica leaf, Aster tataricus, and Glycyrrhiza uralensis. These herbs work synergistically to relieve phlegm, cough, and asthma, and can be used for cough and phlegm symptoms caused by various types of pneumonia. As a compound traditional Chinese medicine preparation, preliminary studies have been conducted on its components and fingerprint spectrum. Based on existing standards, its quality control can be comprehensively implemented. However, traditional Chinese medicine compound preparations are complex and diverse. Further in-depth research is needed to understand the mechanisms by which they exert their effects. In light of this, researchers have proposed various theories for the material basis research of traditional Chinese medicine compound preparations, such as the "component structure" theory, the "identification and quality control model of active substances in traditional Chinese medicine based on component knockout / knockin," and the "equivalent component group" theory. However, the establishment of these theories requires fundamental component research. This study will add the determination of effective components based on previous research and conduct further in-depth research on other aspects of previous research to be used for later pharmacological component research. It will increase applicability, simplify procedures, and reduce research costs in all aspects to make it effective for identification and analysis.

[0003] According to the prior art literature search conducted by the applicant, CN114577941A discloses a fingerprint chromatographic detection method for a traditional Chinese medicine for relieving cough and expectoration. The contents disclosed in this patent document are as follows: (1) Preparation of test solution: Take the traditional Chinese medicine, place it in a volumetric flask, dilute with water and bring to the mark, shake well, filter, and set aside; (2) Preparation of reference solution: Weigh the reference standards of apigenin isoglycyrrhizin, isoglycyrrhizin, baicalin, wogonin, baicalein, and ammonium glycyrrhizate, dilute with methanol and bring to the mark, sonicate to aid dissolution, and prepare the reference solution; (3) Chromatographic conditions: Column: octadecylsilane-bonded silica gel as the packing material; mobile phase: acetonitrile A - 0.1% phosphoric acid aqueous solution B; gradient elution conditions: 0-5 min, 2%-5% A; 5-10 min, 5%-10% A ; 10-15 min, 10%-13% A; 15-20 min, 13%-20% A; 20-35 min, 20%-30% A; 35-45 min, 30%-40% A; 45-50 min, 40%-20% A; 50-55 min, 20%-2% A, flow rate: 0.1-1 ml / min, detection column temperature: 20-30℃, detection wavelength: 250 nm, 354 nm; ⑷, fingerprint spectrum establishment: respectively take the test solution of step ⑴ and the reference solution of step ⑵, and use the chromatographic conditions of step ⑶ to determine them. Prepare multiple batches of test solutions using step (1), and determine them according to the chromatographic conditions of step (3) to obtain multiple batch fingerprint spectra. Import them into the Chinese medicine fingerprint spectrum similarity evaluation software and perform similarity evaluation.Patent document CN116754659A describes a quality detection method for traditional Chinese medicine compound prescriptions based on fingerprint spectroscopy. The chromatographic gradient elution conditions for this method are: A is a 0.4% (v / v) formic acid aqueous solution, B is acetonitrile; 0–5 min, the volume percentage of mobile phase B increases from 2% to 6%; 5–10 min, the volume percentage of mobile phase B increases from 6% to 8%; 10–15 min, the volume percentage of mobile phase B increases from 8% to 15%. %; 15–20 min, the volume percentage of mobile phase B increases from 15% to 20%; 20–30 min, the volume percentage of mobile phase B increases from 20% to 23%; 30–40 min, the volume percentage of mobile phase B increases from 23% to 32%; 40–60 min, the volume percentage of mobile phase B increases from 32% to 35%; 60–80 min, the volume percentage of mobile phase B increases from 35% to 50%; 80–85 min The volume percentage of mobile phase B increased from 50% to 80% at 85–90 min; from 90–95 min, the volume percentage of mobile phase B increased from 80% to 98%; from 90–95 min, the volume percentage of mobile phase B decreased from 98% to 60%; from 95–100 min, the volume percentage of mobile phase B decreased from 60% to 35%; and from 100–105 min, the volume percentage of mobile phase B decreased from 35% to 2%. This method detected 20 chromatographic peaks related to anti-inflammation. Thirteen components were identified, specifically 5,7-dihydroxy-6-methoxyflavonoid-7-O-glucuronide, sucrose, glycyrrhizin-4'-O-β-D-glucose-(1→6)-O-β-D-glucosinolate, glycyrrhizin, norbaicalin-7-O-β-D-glucuronide, baicalin, magnoflorine, 5,7,8-trihydroxyflavonoid-7-O-glucosinolate, baicalin, berberine, wogonin, berberine, and ammonium glycyrrhizate.

[0004] However, the existing methods for detecting the quality components mentioned above have many defects and problems. For example, the detection method in CN114577941A has problems such as long detection time, poor stability of sample solution, and failure to fully characterize the effective components of traditional Chinese medicine. Therefore, it is very important to develop an efficient method for identifying and detecting the effective components in cough syrup. Summary of the Invention

[0005] This invention provides a method for detecting the content of multiple indicators of components in cough syrup oral liquid. This method establishes a method for determining the content of nine key pharmacodynamic components: ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin, isoglycyrrhizin, baicalin, scutellarin A, wogonin, baicalein, and wogonin. This method has the advantages of being simple to operate, highly stable, specific, and practical, and can be used as a method for detecting and evaluating the intrinsic quality components in cough syrup oral liquid production, showing good market application prospects.

[0006] The technical solution of this invention patent application is as follows: A method for detecting the content of multiple components in a cough syrup oral solution, the method comprising the following steps: (1) Preparation of reference solution: Weigh ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin isoglycyrrhizin, isoglycyrrhizin, baicalin, scutellarin A, baicalin, wogonin, and wogonin reference standards, dilute with methanol and bring to volume, sonicate to aid dissolution, and prepare the reference solution. (2) Preparation of test solution: Take cough syrup, place it in a volumetric flask, dilute with methanol and bring to volume to the mark, shake well, filter, and the solution is ready; (3) Chromatographic conditions: The chromatographic column was C18; the mobile phase was methanol (A) - 0.1% phosphoric acid aqueous solution (B); the gradient elution conditions were: 0~14 min, 8% (A); 14~15 min, 8%~40% (A); 15~35 min, 40% (A); 35~40 min, 40%~50% (A); 40~45 min, 50% (A); 45~55 min, 50%~70% (A); 55~60 min, 70%~8% (A); the detection wavelengths were: Channel A, 210 nm, 280 nm, 0~25 min, 210 nm; 25.01~60 min, 280 nm; Channel B, 354 nm; the chromatographic mobile phase flow rate was 0.1~1 mL / min; and the column temperature was 25~35°C. (4) Take the reference solution from step (1) and the test solution from step (2), and determine the content of each active ingredient according to the chromatographic conditions in step (3) above.

[0007] Preferably, the concentrations of the reference solution in step (1) of the detection method are 545 μg / mL, 602.792 μg / mL, 489.02 μg / mL, 462.387 μg / mL, 500.628 μg / mL, 532.14 μg / mL, 591 μg / mL, 521 μg / mL, and 517.44 μg / mL.

[0008] Preferably, the chromatographic column model for step (3) of the detection method is: supperfex JX.

[0009] Preferably, the chromatographic column specifications for step (3) of the detection method are: 4.6 × 250 mm, 5.0 μm.

[0010] Preferably, in step (3) of the detection method, the mobile phase of the 0.1% phosphoric acid aqueous solution contains 0.1% triethylamine.

[0011] Preferably, in step (3) of the detection method, the detection wavelength is 210 nm for detecting ephedrine hydrochloride and pseudoephedrine hydrochloride, 280 nm for detecting baicalin, scutellarin A, baicalein, wogonin, and wogonin, and 354 nm for detecting apigenin and isoliquiritigenin.

[0012] Preferably, in step (3) of the detection method, the flow rate of the chromatographic mobile phase is 1 mL / min and the column temperature is 30°C; Preferably, the detection method can be used for the quality component detection, content detection component research of cough syrup oral liquid, and the analysis and identification of traditional Chinese medicine components.

[0013] To further illustrate the inventiveness of the detection method for the cough syrup oral liquid of the present invention, the experimental contents of the screening of the optimal process parameters of the technical solution of the present invention are summarized as follows.

[0014] Previous studies on flavonoids in Scutellaria baicalensis using different solvents revealed that extraction with methanol (50% or higher) resulted in better stability than extraction with aqueous solution. Stability studies of methanol- and water-soluble baicalin reference standards were also conducted. Tests showed that both methanol- and water-soluble baicalin remained relatively stable within 24 hours. On day 7, under refrigeration, the results for both methanol- and water-soluble baicalin were stable compared to day 1. However, at room temperature, aqueous baicalin underwent hydrolysis, producing baicalein. To ensure the long-term stability of this formulation during the study, methanol was used for dilution and volume adjustment.

[0015] 1. Examination of the research content of this invention The basic requirements for the following examination content are: Preparation of the test sample: Take 1 mL of cough syrup oral solution, place it in a 20 mL volumetric flask, dilute with water and bring to the mark, shake well, filter, and set aside. Chromatographic conditions: Agilent XDB-C18 column (4.6 × 250 mm, 5 μm); column temperature: room temperature; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 210, 280 nm.

[0016] 1.1 Investigation of mobile phase gradient Table 1. Conditions for Exploring Chromatographic Mobile Phase 1.2 Investigation of the mobile phase The effects of methanol-0.1% formic acid solution, methanol-0.1% phosphoric acid solution, methanol-0.1% phosphoric acid solution (containing 0.1% triethylamine), and methanol-0.1% phosphoric acid solution (containing 0.05% triethylamine) on the separation efficiency were investigated respectively. The results are attached. Figure 1(210nm), (280nm): The baseline of formic acid water was uneven, so it was not considered. Phosphoric acid water had a flat baseline, but some peaks showed tailing. Adding 0.05% and 0.1% triethylamine tailing agent improved the peak shape. The results showed that the peak shape improved with increasing triethylamine concentration. (See attached figure). Figure 2 While the overall chromatographic peak shifted, the peak shape and resolution remained unaffected. Therefore, methanol-0.1% phosphoric acid solution (containing 0.1% triethylamine) was used as the analytical mobile phase.

[0017] 1.3 Wavelength Investigation A comparative analysis of 210nm, 254nm, 280nm, and 354nm was conducted, as shown in the appendix. Figures 3-4 The results showed two chromatographic peaks at 210 nm compared to other wavelengths, occurring between 10 and 14 minutes. Comparing 254 nm and 280 nm, the overall number and shape of peaks were consistent, but the peak at 280 nm was generally higher than the peak at 250 nm; therefore, 280 nm was chosen. At 354 nm, the main peak appeared before 36 minutes, with two chromatographic peaks appearing. Therefore, 210, 280, and 354 nm were selected as the detection wavelengths.

[0018] 1.4 Different chromatographic columns The following chromatographic columns were compared: ① Diamonsil 250×4.6mm 5µm ② Ultimate AC-C18 250×4.6mm 5µm ③ Agilent 5 TC-C18(2) 250×4.6mm 5µm ④ Superfex JX-C18 250×4.6mm 5µm ⑤ Agilent XDB-C18 250×4.6mm 5µm. The results were obtained from the attached... Figure 5 It can be seen that the Yuexu and Dima column models have strong elution capabilities, which are not conducive to this study and are therefore excluded. Comparing the Agilent 5 TC-C18(2), Agilent XDB-C18, and Superfex JX-C18, it can be seen that all three columns can be used for this study. However, the retention time of the Agilent 5 TC-C18(2) peak varies significantly, so it is excluded. Further comparison of the Agilent XDB-C18 and Superfex JX-C18 is shown in the attached... Figure 6 It is known that the latter column separation is superior to the former, so the Superfex JX-C18 is preferred for the analysis and detection of cough syrup under these conditions.

[0019] 1.5 Investigation at different column temperatures Comparative analyses were performed at 30℃, 28℃, and 32℃, respectively. See the attached chromatogram for details. Figure 7The detection chromatograms at 210nm and 280nm are attached. Figure 8 The chromatogram is shown at 354 nm. As the temperature gradually increases, the overall retention time of the chromatographic peaks shifts forward. The overall number and resolution of the chromatographic peaks are good at different temperatures, and no peak disruption occurs. At 28°C, the baseline shift is normal, as the peak shift at this temperature occurs at the same time as the wavelength change and elution time in this detection channel at 30 min, resulting in an upward shift of the baseline. Adjusting the temperature too high or too low may affect the detected components and the chromatographic column. Therefore, temperatures below 32°C or room temperature can be used for this study, but 30°C was chosen as the detection temperature based on applicability and column robustness.

[0020] 1.6 Investigation of Flow Velocity Flow rates of 0.8 mL / min, 0.98 mL / min, 1 mL / min, and 1.02 mL / min were compared and analyzed. (See attached...) Figure 9 (210nm, 280nm), Appendix Figure 10 As shown at (354nm), the overall chromatographic peak retention time shifts later as the flow rate decreases. At 0.8 mL / min, the detection time exceeds 60 min. At flow rates of 0.98 mL / min, 1 mL / min, and 1.02 mL / min, the overall retention time and baseline do not change significantly. To ensure that the instrument and detection method can be used for analysis even when fluctuations occur within a certain flow rate range, 1 mL / min is selected as the detection flow rate.

[0021] 1.7 Investigation of Preparation Methods Method 1: Take 2 mL of cough syrup oral solution, add it to a neutral alumina column (100~200 mesh, 2g, inner diameter 1cm), elute with 50% methanol, place in a 25 mL volumetric flask, then add 50% methanol to the mark, shake well, and filter.

[0022] Method 2: Take 1 mL of cough syrup and place it in a 25 mL volumetric flask. Add methanol to bring the volume to the mark, shake well, filter, and set aside.

[0023] This study compares the two methods mentioned above; see appendix for details. Figure 11Method 1 was investigated by varying column loading, elution time, packing material volume, and solvent ratio. It was found that the first few factors had no significant impact on the number and shape of chromatographic peaks. However, repeatability analysis revealed that this method places high demands on both the chromatographic column and operator skill. Compared to Method 1, Method 2 is simpler and more operable, and it elutes most compounds after column chromatography. Method 2 also retains a large number of columns under the same chromatographic conditions, while requiring less stringent standards than Method 1. Furthermore, subsequent specificity and methodological validation demonstrated the stability and feasibility of Method 2; therefore, Method 2 was selected as the sample preparation method.

[0024] The beneficial effects of the patented technical solution of this invention are as follows: (1) Compared with the closest prior art patent (ZL202210224429.3), this invention has several optimizations and improvements, mainly: ① Shortened detection time: The existing technical solution is converted into ordinary liquid chromatography for determination, with a detection time of 132 minutes, while the detection time of this invention is 60 minutes. ② Wider applicability and greater versatility: In particular, the Waters H-class ultra-high performance liquid chromatography instrument used in the closest prior art is expensive and not commonly used in actual applications in pharmaceutical companies. The Waters e-2695 instrument used in this study is more common in pharmaceutical companies and has advantages in terms of detection costs, consumables, and utilization rate in later applications.

[0025] (2) Through extensive experimentation and trial, the present invention has obtained the optimal chromatographic conditions: the mobile phase is methanol (A) - 0.1% phosphoric acid aqueous solution (B); the gradient elution conditions are: 0~14 min, 8% (A); 14~15 min, 8%~40% (A); 15~35 min, 40% (A); 35~40 min, 40%~50% (A); 40~45 min, 50% (A); 45~55 min, 50%~70% (A); 55~60 min, 70%~8% (A); the detection wavelengths are: channel A, 210nm, 280nm, 0~25min, 210nm; 25.01~60min, 280nm; channel B, 354nm.

[0026] (3) The detection method of the present invention also has the following advantages: it simplifies the process, reduces time, and reduces solvent consumption. According to the national drug standard for cough syrup [WS-11419(ZD-1419)-2002-2013Z], ephedrine hydrochloride is determined by acidification, multiple extractions, and evaporation. In this experiment, the determination is carried out directly after volume adjustment, which greatly shortens the sample preparation time and process and makes the determination more convenient. The detection method of the present invention also integrates and simplifies the testing process. In the experimental study, multiple indicators and multiple detections are integrated into one injection and one method, which optimizes resource allocation and process.

[0027] (4) This invention establishes the determination of the content of nine key pharmacodynamic components: ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin, isoglycyrrhizin, baicalin, scutellarin A, wogonin, baicalein, and wogonin. This study adds ephedrine hydrochloride and pseudoephedrine hydrochloride as effective components in the antitussive and expectorant properties of ephedra; apigenin and isoglycyrrhizin as effective anti-inflammatory and antiviral components in licorice; and baicalin and baicalein as effective components in the heat-clearing properties of scutellaria. These effective components are related to the efficacy of the cough syrup oral liquid of this invention and can be used for the intrinsic quality evaluation of this traditional Chinese medicine compound product, further ensuring its intrinsic quality.

[0028] (5) Precision testing results show that the RSD values ​​of the peak areas of the nine components in the cough syrup established in this invention—ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin isoglycyrrhizin, isoglycyrrhizin, baicalin, scutellarin A, baicalein, wogonin, and wogonin—are 2.23%, 1.71%, 0.91%, 0.87%, 1.23%, 1.03%, 1.05%, 0.99%, and 1.07%, respectively. Stability results show that the RSD values ​​of the peak areas of the nine components are 2.69%, 2.72%, 2.81%, 2.42%, 1.14%, 1.08%, 1.09%, 1.25%, and 1.25%, respectively, all less than 3%, indicating good stability of the sample solution within 24 hours. Repeatability tests showed that the peak areas of the nine components had RSD values ​​of 2.64%, 1.86%, 2.52%, 2.98%, 2.37%, 2.64%, 2.52%, 2.88%, and 2.73%, respectively, all less than 3%, indicating good repeatability. Recovery results showed that the average recovery rate of each reference standard ranged from 98% to 103%, with an RSD (n=6) < 3%, indicating good accuracy of the method. Attached Figure Description

[0029] Figure 1 The chromatograms of the test solution (210 nm, 280 nm) were obtained by examining the mobile phase. Figure 2 The chromatogram of the test solution (354 nm) was examined using the mobile phase. Figure 3 Chromatograms of the test solution examined at wavelengths of 210 nm, 254 nm, and 280 nm. Figure 4 The chromatogram of the test solution examined at a wavelength of 354 nm; Figure 5 Chromatograms of the test solution examined on different chromatographic columns (210 nm, 280 nm). Figure 6 Chromatograms of the test solution (354 nm) examined using different chromatographic columns. Figure 7 Chromatograms of the test sample solution at different column temperatures (210 nm, 280 nm); Figure 8 Chromatograms of the test solution at different column temperatures (354 nm); Figure 9 Chromatograms of the test solution at flow rates (210 nm, 280 nm); Figure 10 Chromatogram of the test solution at 354 nm for flow rate determination; Figure 11 Chromatograms of test sample solutions prepared by different methods were examined. Figure 12 Chromatograms of cough syrup oral solution and reference solution (A) 210nm and 280nm chromatograms, where a is the reference solution and b is the test solution. 1. Ephedrine hydrochloride 2. Pseudoephedrine hydrochloride 3. Isoglycyrrhizin 4. Isoglycyrrhizin 5. Baicalin 6. Phytosine A glycoside 7. Baicalin 8. Baicalein 9. Baicalein; Figure 13 Chromatograms of cough syrup oral solution and reference solution (B) at 354 nm; Figure 14 Negative control chromatogram A: Negative control chromatograms of Scutellaria baicalensis and Ephedra sinica; Figure 15 Chromatogram B, negative control chromatogram of licorice; Figure 16 Chromatogram of Scutellaria baicalensis solution; Figure 17 Chromatogram of ephedra solution; Figure 18 Chromatogram of licorice herb solution. Detailed Implementation

[0030] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0031] Example 1: Construction of the multi-index component content detection method for cough syrup oral liquid of the present invention 1. Instruments and reagents 1.1 Instruments and Reagents Waters e 2695 high performance liquid chromatograph (Waters Corporation, USA); KUDOU ultrasonic instrument (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); MS430TS electronic balance (Mettler-Toledo Instruments Shanghai Co., Ltd.); XPE205 electronic balance (Mettler-Toledo Instruments Shanghai Co., Ltd.); Merck Milli-Q pure water system (Merck Chemical Technology Shanghai Co., Ltd.).

[0032] Methanol, acetonitrile [GR, Thermo Fisher Scientific (China) Co., Ltd.]; methanol (AR, China National Chemical Reagent Co., Ltd.), phosphoric acid (AR, Hongyan Reagent Factory, Hedong District, Tianjin), triethylamine (AR, Luoyang Haohua Chemical Reagent Co., Ltd.).

[0033] 1.2 Materials The following products were purchased from Lemite Pharmaceuticals: A-7-O-β-D-glucuronide (batch number: DSTDQ004001, purity ≥98%); apigenin isoglycyrrhizin (batch number: DST241028-166, purity ≥98%); isoglycyrrhizin (batch number: MUST-24111310, purity ≥99.31%); baicalin (batch number: 110715-202223, purity ≥93.3%); and wogonin (batch number: 112002-202303, purity ≥99%). 0.06%); Baicalein (batch number: 11595-202309, purity ≥98.6%); Baicalein (batch number: 111514-202207, purity ≥99.06%); Ephedrine hydrochloride (batch number: 171241-202310, purity ≥99.8%); Pseudoephedrine hydrochloride (batch number: 171237-202211, purity ≥99.8%), China National Institutes for Food and Drug Control; Cough Syrup was provided by Shaanxi Buchang Pharmaceutical Co., Ltd.; Menthol (batch number: X241017) was purchased from Huangshan Tianmu Menthol Pharmaceutical Co., Ltd.; Fritillaria cirrhosa, Eriobotrya japonica leaf, Aster tataricus, Ephedra sinica, Scutellaria baicalensis, Papaver somniferum husk, and Glycyrrhiza uralensis were purchased from Shaanxi Xingshengde Pharmaceutical Co., Ltd.

[0034] 2. Methods and Results 2.1 Chromatographic conditions The chromatographic column was a Superfex JX-C18 (4.6 × 250 mm, 5.0 μm); the mobile phase was methanol (A) - 0.1% phosphoric acid aqueous solution (containing 0.1% triethylamine) (B); the gradient elution conditions were: 0–14 min, 8% (A); 14–15 min, 8%–40% (A); 15–35 min, 40% (A); 35–40 min, 40%–50% (A); 40–45 min, 50% (A); 45–55 min, 50%–70% (A); 55–60 min, 70%–8% (A); column temperature: room temperature; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: channel A, 210 nm, 280 nm (0–25 min, 210 nm; 25.01–60 min, 280 nm); channel B, 354 nm. Ephedrine hydrochloride, pseudoephedrine hydrochloride 210 nm, baicalin, scutellarin A, baicalein, wogonin, wogonin 280, apigenin isoglycyrrhizin, isoglycyrrhizin 354 nm.

[0035] 2.2 Solution Preparation 2.2.1 Preparation of the reference solution Accurately weigh appropriate amounts of ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin isoglycyrrhizin, isoglycyrrhizin, baicalin, scutellarin A, baicalein, wogonin, and wogonin reference standards. Dilute with methanol to the mark and sonicate to aid dissolution. Prepare reference solutions with concentrations of 545 μg / mL, 602.792 μg / mL, 489.02 μg / mL, 462.387 μg / mL, 500.628 μg / mL, 532.14 μg / mL, 591 μg / mL, 521 μg / mL, and 517.44 μg / mL, respectively. Detect according to the chromatographic conditions in 3.1. Refer to the chromatogram for the results. Figure 12-13 .

[0036] 2.2.2 Preparation of the test solution Take 1 mL of the cough syrup oral solution and place it in a 5 mL or 20 mL volumetric flask. Dilute with methanol and bring to the mark. Shake well, filter, and set aside. Detect according to the chromatographic conditions in 3.1. Refer to the chromatogram for the results. Figure 12-13 .

[0037] 2.2.3 Preparation of Aqueous Decoction Solution Referring to the national drug standard for cough syrup [WS-11419(ZD-1419)-2002-2013Z], the following eight medicinal materials are used: Fritillaria cirrhosa, Eriobotrya japonica leaf, Aster tataricus, Ephedra sinica, Scutellaria baicalensis, Papaver somniferum husk, Glycyrrhiza uralensis, and Menthol. Except for Menthol, Ephedra sinica is steam distilled, and the distillate and medicinal liquid are collected for later use. The residue is decocted twice with the remaining six medicinal materials, including Fritillaria cirrhosa, for 2 hours the first time and 1.5 hours the second time. The decoctions are combined, filtered, and the filtrate is combined with the medicinal liquid after extracting the volatile oil. The mixture is concentrated to a clear paste with a relative density of 1.10-1.15 (50℃). Ethanol is added to make the alcohol content 60%, and the mixture is allowed to stand for 24 hours. The mixture is then filtered, the ethanol is recovered from the filtrate, and the mixture is concentrated to a clear paste with a relative density of 1.20-1.25 (50℃). The above distillate and Menthol are then added to obtain the final product.

[0038] 2.2.4 Preparation of negative solution (1) Ephedra negative: Refer to the national drug standard for cough syrup to prepare ephedra-deficient negative solutions.

[0039] (2) Scutellaria baicalensis negative: Refer to the national drug standard for cough syrup and prepare negative solutions for those lacking Scutellaria baicalensis.

[0040] (3) Licorice negative: Refer to the national drug standard for cough syrup and prepare licorice-deficient negative solutions respectively.

[0041] 2.2.5 Preparation of herbal solution Scutellaria baicalensis: Prepare the test solution of Scutellaria baicalensis slices according to the 2025 edition of the Chinese Pharmacopoeia. The baicalin content shall not be less than 8%.

[0042] Ephedra: Prepare a test solution of ephedra medicinal material according to the 2025 edition of the Chinese Pharmacopoeia, containing a total amount of ephedrine hydrochloride and pseudoephedrine hydrochloride of not less than 0.8%.

[0043] Licorice: Prepare a licorice decoction sample solution according to the 2025 edition of the Chinese Pharmacopoeia. The solution should contain not less than 0.5% glycyrrhizin and not less than 2.0% glycyrrhizic acid.

[0044] 2.3 Methodological Validation 2.3.1 Specificity assessment and chromatographic peak attribution analysis The negative solution was prepared according to "2.2.4" and analyzed under the chromatographic conditions described in "2.1". The following chromatogram was obtained. See chromatogram for details. Figure 14-18 As shown in the following figure, ephedrine hydrochloride and pseudoephedrine hydrochloride belong to ephedra medicinal materials, and other medicinal materials do not interfere with these two components. Apigenin and isoliquiritin belong to licorice medicinal slices, and other medicinal materials do not interfere with these two components. Baicalin, scutellarin A, wogonin, baicalein, and wogonin belong to scutellaria medicinal materials, and other medicinal materials do not interfere with these five components.

[0045] 2.3.2 Preparation of Standard Curve Accurately pipette appropriate amounts of each reference solution into volumetric flasks, dilute with methanol and bring to volume to the mark, shake well to obtain a series of linear reference solutions, inject them under the chromatographic conditions in section “2.1”, plot the standard curve with peak area Y as the ordinate and concentration X (μg / mL) as the abscissa, calculate the regression equation, and the results are shown in Table 2.

[0046] Table 2 Standard curves and linear ranges of the nine indicator components Reference Standard curve Concentration range (μg / mL) Ephedrine hydrochloride y=21071x-27991 4.36~109 0.9995 pseudoephedrine hydrochloride y = 21615x - 990.5 2.411~60.279 0.9995 celery isoliquiritigenin y = 25165x - 2697.2 0.925~23.119 0.9991 Isoliquiritin y=39057x-15512 0.978~24.451 0.9994 baicalin y=35265x-159288 20.027~500.628 0.9995 Thousand-layer paper pigment A glycoside y = 29412x + 9229.3 2.129~53.214 0.9999 baicalin y = 36679x + 9342.4 5.174~129.36 0.9994 Baicalein y = 54078x + 23563 2.364~39.1 0.9993 baicalein y = 54867x + 10137 2.084~52.1 0.9994 2.3.3 Precision Examination Accurately pipette appropriate amounts of each reference standard, mix them, and inject them six times consecutively under the chromatographic conditions specified in section "2.1". Calculate the RSD values ​​of the peak areas of the nine components—ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin isoglycyrrhizin, isoglycyrrhizin, baicalin, scutellarin A, baicalein, wogonin, and wogonin—to be 2.23%, 1.71%, 0.91%, 0.87%, 1.23%, 1.03%, 1.05%, 0.99%, and 1.07%, respectively. This indicates that the instrument has good precision.

[0047] 2.3.4 Stability Assessment Take the cough syrup oral solution (batch number: 210226), prepare the test solution according to the "2.2.2" section, and inject it at 0, 2, 4, 6, 8, 10, 12, and 24 h under the chromatographic conditions of the "2.1" section. The peak areas of ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin isoglycyrrhizin, isoglycyrrhizin, baicalin, scutellarin A, baicalein, wogonin, and wogonin were calculated, and the RSD values ​​of the sample solution were 2.69%, 2.72%, 2.81%, 2.42%, 1.14%, 1.08%, 1.09%, 1.25%, and 1.25%, respectively. All of these values ​​were less than 3%, indicating that the sample solution had good stability within 24 h.

[0048] 2.3.5 Repeatability Test Six test solutions were prepared in parallel according to the same batch of cough syrup oral liquid, following the procedure in section "2.2.2". The solutions were then analyzed under the chromatographic conditions in section "2.1". The peak areas of the nine components were measured sequentially, and the RSD values ​​were 2.64%, 1.86%, 2.52%, 2.98%, 2.37%, 2.64%, 2.52%, 2.88%, and 2.73%, respectively, all less than 3%. This method has good repeatability.

[0049] 2.3.6 Recovery rate analysis Take 6 portions each of 0.5 mL and 1 mL of the same batch of cough syrup (batch number: 210226) with known content, and accurately add each reference solution. Prepare the test solution under the chromatographic conditions in section "2.2.2". Measure the solution under the chromatographic conditions in section "2.1" and calculate the recovery rate. The average recovery rate of each reference standard ranged from 98% to 103%, and its RSD (n=6) was <3%, as shown in Table 3 below, indicating that the method has good accuracy.

[0050] Table 3 Results of the recovery test of the reference standard Reference Sample concentration (μg / mL) Reference standard added (μg / mL) Total measured amount (μg / mL) Average recovery rate (%) RSD (%) Ephedrine hydrochloride 211.486 254.000 463.833 99.35% 2.45% pseudoephedrine hydrochloride 117.809 120.558 236.786 98.69% 1.48% celery glycyrrhizin 18.121 15.893 33.786 98.56% 0.76% Isoliquiritin 42.493 41.615 83.824 99.32% 1.76% baicalin 1004.061 801.091 1582.376 98.76% 1.65% Layer-rich protein A glycoside 113.079 125.171 240.195 101.55% 1.96% baicalin 245.930 254.800 494.779 103.16% 1.63% Baicalein 66.796 76.830 141.537 97.28% 0.91% baicalein 39.857 36.470 75.950 98.97% 1.50% In summary, the method established in this invention for determining the content of nine components—ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin isoglycyrrhizin, isoglycyrrhizin, baicalin, scutellarin A, baicalein, wogonin, and wogonin—has the advantages of good stability, high reproducibility, and simple operation. This detection method can be used as an internal control quality testing method in the intrinsic quality and production inspection of cough syrup oral liquid, and has broad market application prospects for the evaluation of the intrinsic quality of this traditional Chinese medicine compound.

Claims

1. A method for detecting the content of multiple components in a cough syrup, characterized in that, The detection method includes the following steps: (1) Preparation of reference solution: Weigh ephedrine hydrochloride, pseudoephedrine hydrochloride, apigenin isoglycyrrhizin, isoglycyrrhizin, baicalin, scutellarin A, baicalin, wogonin, and wogonin reference standards, dilute with methanol and bring to volume, sonicate to aid dissolution, and prepare the reference solution. (2) Preparation of test solution: Take cough syrup, place it in a volumetric flask, dilute with methanol and bring to volume to the mark, shake well, filter, and the solution is ready; (3) Chromatographic conditions: The chromatographic column was C18; the mobile phase was methanol (A) - 0.1% phosphoric acid aqueous solution (B); the gradient elution conditions were: 0~14 min, 8% (A); 14~15 min, 8%~40% (A); 15~35 min, 40% (A); 35~40 min, 40%~50% (A); 40~45 min, 50% (A); 45~55 min, 50%~70% (A); 55~60 min, 70%~8% (A); the detection wavelengths were: Channel A, 210 nm, 280 nm, 0~25 min, 210 nm; 25.01~60 min, 280 nm; Channel B, 354 nm; the chromatographic mobile phase flow rate was 0.1~1 mL / min; and the column temperature was 25~35°C. (4) Take the reference solution from step (1) and the test solution from step (2), and determine the content of each active ingredient according to the chromatographic conditions in step (3) above.

2. The detection method as described in claim 1, characterized in that, The concentrations of the reference solutions in step (1) of the detection method are 545 μg / mL, 602.792 μg / mL, 489.02 μg / mL, 462.387 μg / mL, 500.628 μg / mL, 532.14 μg / mL, 591 μg / mL, 521 μg / mL, and 517.44 μg / mL, respectively.

3. The detection method as described in claim 1, characterized in that, The detection method step (3) chromatographic column model: supperfex JX.

4. The detection method as described in claim 1, characterized in that, The detection method step (3) chromatographic column specifications: 4.6×250 mm, 5.0 μm.

5. The detection method as described in claim 1, characterized in that, The detection method step (3) contains 0.1% triethylamine in the mobile phase of 0.1% phosphoric acid aqueous solution.

6. The detection method as described in claim 1, characterized in that, The detection method step (3) uses the following wavelengths: 210 nm for detecting ephedrine hydrochloride and pseudoephedrine hydrochloride; 280 nm for detecting baicalin, scutellarin A, baicalein, wogonin, and wogonin; and 354 nm for detecting apigenin and isoglycyrrhizin.

7. The detection method as described in claim 1, characterized in that, In step (3) of the detection method, the flow rate of the chromatographic mobile phase is 0.3 mL / min, and the column temperature is 30°C.

8. The detection method as described in claims 1 to 7, characterized in that, The detection method described herein can be used for the quality component detection and content analysis of cough syrup oral liquid, as well as for the analysis and identification of traditional Chinese medicine components.

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