Multi-index component content and fingerprint spectrum detection method of Shenxian Shengmai oral liquid

Abstract

The application provides a multi-index component content and fingerprint spectrum detection method of Shenshen Shengmai oral liquid, the fingerprint spectrum of the Shenshen Shengmai oral liquid is established by adopting HPLC wavelength switching method, and the contents of 10 important active components, i.e. danshensu, pseudoephedrine hydrochloride, bakuchiol glycoside, isobakuchiol glycoside, ferulic acid, rosmarinic acid, ginsenoside Rg1, salvianolic acid B, bakuchiol, isobakuchiol, and epimediumin C and icariin are determined, so that the quality of the Shenshen Shengmai oral liquid can be comprehensively and systematically identified, the quantitative analysis of the multi-index components is realized, and a more comprehensive scientific basis for effectively controlling the quality of the Shenshen Shengmai oral liquid is provided.

Description

Technical Field

[0001] This invention relates to the field of chemical detection and analysis of traditional Chinese medicine, and more specifically to a method for detecting the content of multiple components and their fingerprint spectrum in Shenxian Shengmai oral liquid. Background Technology

[0002] Shenxian Shengmai Oral Liquid is a traditional Chinese medicine compound preparation made from eight medicinal herbs: red ginseng, epimedium, psoralea (salt-processed), wolfberry, ephedra, asarum, salvia miltiorrhiza, and leech. This medicine is a product of Shandong Buchang Pharmaceutical Co., Ltd. Its main functions are to warm and tonify the heart and kidneys, and promote blood circulation and remove blood stasis. This product is used for Yang deficiency with slow pulse, characterized by slow, knotted pulse, palpitations, chest tightness, aversion to cold, cold limbs, soreness and weakness of the lower back and knees, shortness of breath, fatigue, or dizziness; a pale tongue or tongue with teeth marks, or petechiae or ecchymosis on the tongue. It is equivalent to mild to moderate sinus bradycardia and mild sick sinus syndrome without supraventricular tachyarrhythmia due to heart and kidney Yang deficiency and cold stagnation of blood vessels. The applicant has filed multiple invention patent applications for this product: patent application numbers are 200710019161.5, 200830117798.3, 201010275244.2, 202010831085.3, and 202210879150.9. The patent protection topics mainly focus on pharmaceutical compositions for treating arrhythmias, preparation processes, detection methods for volatile components, detection methods, ultra-high performance liquid chromatography detection methods, and applications in drugs for preventing and treating diabetic complications.

[0003] This drug is clinically used to treat bradyarrhythmias and is classified as a Class III new drug in China. However, due to the relatively large number of medicinal materials and complex chemical composition of Shenxian Shengmai Oral Liquid, a comprehensive and sound quality evaluation system is crucial for its quality control. Existing technical literature reports, such as Yang Juanying, Lin Yuhong, Xie Wei, et al., published in the Shaanxi Journal of Traditional Chinese Medicine on September 5, 2011, "HPLC Fingerprint Study of Shenxian Shengmai Oral Liquid," but it only detected and identified a limited number of characteristic peaks, identifying only three components: ginsenosides Rb, Rg, and icariin. No further research reports on the determination of the content of other indicator components were found, failing to comprehensively reflect the overall quality of Shenxian Shengmai Oral Liquid. Chromatographic fingerprinting of traditional Chinese medicine can express complete material information of traditional Chinese medicine and characterize the overall properties of chemical components. It is a classic and effective method for qualitatively identifying the authenticity, quality, consistency, and stability of traditional Chinese medicine materials and their preparations. Combining traditional Chinese medicine fingerprinting with quantitative analysis of multiple indicator components, its application in the quality control of traditional Chinese medicine can achieve both qualitative and quantitative evaluation, ensuring the stability of traditional Chinese medicine quality and its safe and controllable clinical application. This study used HPLC to establish the fingerprint spectrum of Shenxian Shengmai oral liquid and simultaneously determined the content of 10 index components, providing a scientific basis for its comprehensive and systematic quality control. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting the content of multiple components and their fingerprint chromatograms in Shenxian Shengmai oral liquid. This invention establishes a fingerprint chromatogram of Shenxian Shengmai oral liquid using HPLC wavelength switching method, and simultaneously determines the content of 10 important active ingredients, including tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, ferulic acid, rosmarinic acid, ginsenoside Rg1, salvianolic acid B, psoralen, isopsoralen, asparagine C, and icariin. This method can comprehensively and systematically identify the quality of Shenxian Shengmai oral liquid and achieve quantitative analysis of multiple components, providing a more comprehensive scientific basis for effectively controlling the quality of Shenxian Shengmai oral liquid.

[0005] The technical solution provided by this invention is as follows:

[0006] A method for detecting multiple components of Shenxian Shengmai oral liquid, the method comprising the following steps:

[0007] (1) Mixed reference solution: Weigh out the reference standards of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, ferulic acid, rosmarinic acid, ginsenoside Rg1, salvianolic acid B, psoralen, isopsoralen, cuscuta citrate C and icariin, dissolve and dilute with methanol, mix well, and prepare a mixed reference solution.

[0008] (2) Test solution: Take a sample of Shenxian Shengmai oral liquid, add methanol to dissolve and dilute it, filter it through a microporous membrane, and take the filtrate to obtain the test solution;

[0009] (3) Chromatographic conditions: The chromatographic column was C18, the mobile phase was acetonitrile (A) and 0.2% phosphoric acid aqueous solution (B), gradient elution: 0~15 min, 5% A; 15~25 min, 5%~15% A; 25~40 min, 15%~18% A; 40~45 min, 18%~22% A; 45~95 min, 22%~25% A; 95~100 min, 25%~5% A; the detection wavelength was 210~270 nm, the flow rate was 0.6~1.4 mL•min-1, the column temperature was 25~35°C, and the injection volume was 5~15 µL.

[0010] (4) Chromatographic peak determination: Inject the mixed reference solution and test solution prepared in steps (1) and (2) above into the liquid chromatograph, and determine and record the chromatogram according to the chromatographic conditions in step (3) above.

[0011] Preferably, in the mixed reference solution of step (1), the linear range of the contents of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, rosmarinic acid, salvianolic acid B, psoralen, isopsoralen, cuscutacin C, and icariin is 32.90 ~ 246.67 μg•mL, respectively. -19.60 ~ 71.67 μg•mL -1 57.30 ~ 429.33 μg•mL -1 54.50 ~408.00 μg•mL -1 2.70 ~ 20.40 μg•mL -1 14.70 ~ 110.00 μg•mL -1 7.00 ~ 52.67 μg•mL -1 6.50~48.33μg•mL -1 5.20 ~ 38.67 μg•mL -1 10.70 ~ 80.00 μg•mL -1 .

[0012] Preferably, in step (2), the methanol dilution factor in the test solution is 8 to 20 times, preferably 16 times.

[0013] Preferably, in step (2), the methanol in the test solution is diluted 16 times.

[0014] Preferably, in step (1), the reference solution is mixed, and the chromatographic column is of the Ultimate LP type.

[0015] Preferably, the chromatographic conditions for step (3) are as follows: the detection wavelength is 270 nm, 0 ~ 16 min, 24 ~ 38 min; 210 nm, 16 ~ 24 min, 38 ~ 100 min.

[0016] Preferably, the chromatographic conditions for step (3) are as follows: the detection flow rate is 0.6–1.4 mL•min⁻¹, the column temperature is 25–35°C, and the injection volume is 5–15 µL.

[0017] Preferably, in step (3), the chromatographic conditions are as follows: the detection flow rate is 1 mL•min⁻¹, the column temperature is 30°C, and the injection volume is 10 µL.

[0018] Preferably, in step (4) chromatographic peak determination, 10 batches of test sample solutions are injected for analysis, and the detection data are imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The results are calculated, and the similarity between the test sample solution and the control fingerprint spectrum is ≥0.93.

[0019] To highlight the innovativeness of the technical solution for detecting the content and fingerprint spectrum of the ginseng and shenshengmai oral liquid in this invention, we provide the following partial experimental results selected during this experiment.

[0020] ① Investigation of the preparation method of the test solution

[0021] The original solution of Shenxian Shengmai oral liquid is an aqueous extract. In this experiment, methanol, an organic solvent, was used for extraction and dilution. The solvent ratios of 2, 4, 6, 8, and 16 were investigated. The results showed that when the solvent ratio was 16, the chromatographic peak information was richer, the peak shape was better, and the relative peak area was more suitable. Therefore, methanol, with a volume of 16, was chosen as the extraction solvent.

[0022] ② Optimization of chromatographic conditions

[0023] This experiment investigated different detection wavelengths (203, 210, 220, 230, 245, 254, 270, 280, 290, and 360 nm), and found that the maximum absorption was observed at 210 nm and 270 nm, respectively. Since Shenxian Shengmai Oral Liquid is made from eight kinds of Chinese medicinal materials, the composition is complex and the properties of each component are very different. It is impossible to achieve the ideal separation effect at only one wavelength. Therefore, this experiment used the wavelength conversion function of HPLC to carry out the determination at 210 nm and 270 nm wavelengths at different time intervals. This experiment investigated the different types of mobile phases (methanol-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid) and column temperatures (25 °C, 30 °C, 35 °C, 40 °C). The results showed that when using acetonitrile-0.2% phosphoric acid aqueous solution as the mobile phase and at a column temperature of 30 °C, the chromatographic peak information obtained was the most abundant, the baseline was relatively stable, and the peak shape and resolution were also excellent. Therefore, acetonitrile-0.2% phosphoric acid aqueous solution was finally selected as the mobile phase, and the column temperature was 30 °C.

[0024] Selection of indicator components

[0025] The fingerprint spectrum of Shenxian Shengmai Oral Liquid, after comparison with reference standards, identified 12 peaks. Referring to the quality control indicators of each medicinal material in the prescription composition as specified in the 2020 edition of the Chinese Pharmacopoeia, ginsenoside Rg1 is an indicator component of red ginseng, pseudoephedrine hydrochloride is an indicator component of ephedra, salvianolic acid B is an indicator component of tanshinone, psoralen and isopsoralen are indicator components of psoralea, and icariin C and epimedium are indicator components of epimedium. Furthermore, it is also noteworthy that tanshinone and rosmarinic acid are not only important active components of tanshinone but also potential quality markers for Shenxian Shengmai Oral Liquid; psoralen and isopsoralen are important active components of psoralea, which can be converted into psoralen and isopsoralen under certain conditions, and are also the two components with the highest proportion among all detected components. Therefore, except for ginsenoside Rg1, which is not suitable for accurate quantification under the method established in this experiment, the other 10 components mentioned above are included in the content determination index of Shenxian Shengmai oral liquid to comprehensively reflect the quality of Shenxian Shengmai oral liquid.

[0026] The beneficial effects of the multi-index component content and fingerprint spectrum detection method of this invention are as follows:

[0027] This invention establishes methods for determining the content of 10 indicator components. The linear ranges for the content of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, rosmarinic acid, salvianolic acid B, psoralen, isopsoralen, asparagine C, and icariin are 32.90 ~ 246.67 μg•mL, respectively. -1 9.60 ~ 71.67 μg•mL -1 57.30 ~ 429.33 μg•mL -1 54.50 ~ 408.00 μg•mL -1 2.70 ~ 20.40 μg•mL -1 14.70 ~ 110.00 μg•mL -1 7.00 ~ 52.67 μg•mL -1 6.50 ~ 48.33 μg•mL -1 5.20 ~ 38.67 μg•mL -1 10.70 ~ 80.00 μg•mL -1 The precision test results showed that the RSD of the relative retention time of each common peak was 0.01%~0.36%, and the RSD of the relative peak area was 0.05%~2.67%, indicating good instrument precision. The stability test results showed that the RSDs of the peak areas for tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, rosmarinic acid, salvianolic acid B, psoralen, isopsoralen, ascorbic acid C, and icariin were 1.98%, 1.31%, 1.88%, 1.83%, 1.73%, 0.86%, 2.48%, 2.69%, 1.70%, and 1.82%, respectively. The repeatability test results showed that the RSD of the relative retention time of each common peak was 0.02%~0.27%, and the RSD of the relative peak area was 0.06%~2.88%, indicating that the content detection method of this invention has good precision, stability, and repeatability.

[0028] Using the optimized content detection method of this invention, the content of tanshinone in 10 batches of Shenxian Shengmai oral liquid was determined to be 1.218–2.781 mg·mL. -1 The content of pseudoephedrine hydrochloride is 0.333–0.702 mg / mL. -1 Psoralen content: 3.043–4.120 mg / mL -1 Isopsoralen content: 2.083–3.486 mg / mL -1 Rosmarinic acid content: 0.171–0.203 mg / mL -1The content of salvianolic acid B is 0.158–1.046 mg·mL. -1 Psoralen content: 0.189–0.286 mg / mL -1 Isopsoralen content: 0.196–0.323 mg / mL -1 The content of asparagine C is 0.231–0.403 mg / mL. -1 The content of icariin is 0.323–0.512 mg / mL. -1 The results of the upper and lower limits of the content detection of 10 effective substance indicators in the above 10 batches of Shenxian Shengmai oral liquid show that the content of the above effective indicator components varies little, which can more comprehensively ensure the quality stability of Shenxian Shengmai oral liquid and ensure its clinical efficacy advantage.

[0029] The fingerprint spectrum detection method established in this invention identified 28 peaks, of which 12 were designated. The similarity scores of the fingerprint spectra of 10 batches of samples and the control were calculated to be 0.934, 0.986, 0.994, 0.999, 0.987, 0.991, 0.994, 0.940, 0.995, and 0.991, respectively. The similarity among the 10 batches of samples was all above 0.934. This indicates that the quality of different batches of Shenxian Shengmai oral liquid has good consistency and can be used in the large-scale production and quality inspection of this product, showing good market application prospects. Attached Figure Description

[0030] The accompanying drawings, which are provided to further illustrate the invention and constitute a part of this invention, are illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention.

[0031] Figure 1 - The HPLC fingerprint (A) and HPLC chromatogram of mixed reference standard I (B) of 10 batches of Xianshengmai oral liquid are shown. Among them, the chromatographic peak 2 is tanshinone, the chromatographic peak 5 is pseudoephedrine hydrochloride, the chromatographic peak 9 is psoralen, the chromatographic peak 10 is isopsoralen, the chromatographic peak 11 is ferulic acid, the chromatographic peak 17 is rosmarinic acid, the chromatographic peak 20 is ginsenoside Rg1, the chromatographic peak 21 is salvianolic acid B, the chromatographic peak 24 is psoralen, the chromatographic peak 25 is isopsoralen, the chromatographic peak 27 is icariin C, and the chromatographic peak 28 is icariin.

[0032] Figure 2- HPLC chromatograms of blank control (A), mixed reference standard II (B), and test sample (C), wherein chromatographic peak 1 is tanshinone; chromatographic peak 2 is pseudoephedrine hydrochloride; chromatographic peak 3 is psoralen; chromatographic peak 4 is isopsoralen; chromatographic peak 5 is rosmarinic acid; chromatographic peak 6 is salvianolic acid B; chromatographic peak 7 isopsoralen; chromatographic peak 8 isopsoralen; chromatographic peak 9 is icariin C; and chromatographic peak 10 is icariin. Detailed Implementation

[0033] To provide a more complete understanding of the implementation of this invention, the following description uses typical embodiments to further illustrate the invention. Unless otherwise defined, the technical or scientific terms used in this patent application specification and claims should have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains.

[0034] Example 1: Establishment of the fingerprinting method for Shenxian Shengmai oral liquid of the present invention

[0035] 1. Materials

[0036] 1.1 Instruments

[0037] Agilent 1260 high performance liquid chromatography system (Agilent Technologies); XM-P22H stepless ultrasonic cleaner (Kunshan Xiaomei Ultrasonic Instrument Co., Ltd.); BT25S and BS210S electronic analytical balances (Beijing Sartorius Scientific Instruments Co., Ltd.).

[0038] 1.2 Drugs and Reagents

[0039] Ten batches of Shenxian Shengmai Oral Liquid were selected, numbered S1 to S10, with batch numbers 160210, 181201, 181202, 191001, 210401, 210402, 210403, 211002, 220202, and 220203, respectively, provided by Shaanxi Buchang Pharmaceutical Co., Ltd. Reference standards pseudoephedrine hydrochloride (batch number 171237-201510), salvianolic acid B (batch number 111562-201212), chamomile C (batch number 111780-201905), and ferulic acid (batch number 1100773-201018) were purchased from the National Institutes for Food and Drug Control; tanshinone (batch number H09N10S102463) and rosmarinic acid (batch number Y06A9K67402) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; psoralen... Psoralen (batch number CHB-B-163) and isopsoralen (batch number CHB-Y-280) were purchased from Chengdu Kloma Biotechnology Co., Ltd.; psoralen (batch number 110739-200310), isopsoralen (batch number 0738-200108), ginsenoside Rg1 (batch number 110703-201530), and icariin (batch number 110737-200312) were purchased from the National Institutes for Food and Drug Control, China. The purity of all the above reference standards was >98%. Acetonitrile was chromatographic grade (Thermo Fisher Scientific, USA), water was Wahaha purified water, and other reagents were analytical grade.

[0040] 2. Methods and Results

[0041] 2.1 Chromatographic conditions

[0042] The chromatographic column was Ultimate LP-C. 18 (4.6 mm × 250 mm, 5 µm), the mobile phase was acetonitrile (A) and 0.2% phosphoric acid aqueous solution (B), gradient elution (0–15 min, 5% A; 15–25 min, 5%–15% A; 25–40 min, 15%–18% A; 40–45 min, 18%–22% A; 45–95 min, 22%–25% A; 95–100 min, 25%–5% A), detection wavelength was 270 nm (0–16 min; 24–38 min) and 210 nm (16–24 min; 38–100 min), flow rate was 1.0 mL·min -1 The column temperature was 30 °C and the injection volume was 10 µL.

[0043] 2.2 Solution Preparation

[0044] 2.2.1 Mixed reference solution

[0045] Accurately weigh appropriate amounts of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, ferulic acid, rosmarinic acid, ginsenoside Rg1, salvianolic acid B, psoralen, isopsoralen, asparagine C, and icariin reference standards, and place them separately in 10 mL volumetric flasks. Dissolve and dilute to volume with methanol to prepare stock solutions for each reference standard. Accurately pipette appropriate amounts of each stock solution, vortex to mix, and prepare concentrations of 206.51, 50.00, 374.42, 355.81, 29.77, 35.58, 168.84, 76.74, 36.74, 33.72, 26.98, and 69.77 μg·mL, respectively. -1 A mixed reference solution (I) is prepared for fingerprint spectroscopy.

[0046] Then, accurately pipette appropriate amounts of stock solutions of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, rosmarinic acid, salvianolic acid B, psoralen, isopsoralen, asparagine C, and icariin, respectively, and vortex to prepare solutions with mass concentrations of 246.67, 71.67, 429.33, 408.00, 20.40, 110.00, 52.67, 48.33, 38.67, and 80.00 μg·mL, respectively. -1 A mixed reference solution (II) is provided for the determination of multiple index contents.

[0047] 2.2.2 Test solution

[0048] Accurately pipette a certain amount of Shenxian Shengmai oral liquid, add 16 times the volume of methanol to dissolve and dilute it, filter it through a 0.45 μm microporous membrane, and collect the filtrate to obtain the product.

[0049] 2.3 Establishment of HPLC fingerprint chromatogram

[0050] 2.3.1 Precision Test

[0051] Take the same sample solution (S10) and inject it six times consecutively under the chromatographic conditions described in section "2.1", recording the chromatograms. Using psoralen (peak 9) as the reference peak, the RSD of the relative retention time of each common peak was calculated to be 0.01% to 0.36%, and the RSD of the relative peak area was 0.05% to 2.67%, indicating good instrument precision.

[0052] 2.3.2 Stability Test

[0053] Take the same sample solution (S10) and perform the determination at room temperature for 0, 2, 4, 8, 12 and 24 h under the chromatographic conditions in section “2.1”. With psoralen (peak 9) as the reference peak, the RSD of the relative retention time of each common peak was calculated to be 0.02% ~ 0.48%, and the RSD of the relative peak area was 0.07% ~ 2.84%, indicating that the sample is stable at room temperature for 24 h.

[0054] 2.3.3 Repeatability Test

[0055] Six test solutions were prepared in parallel using the same batch of Shenxian Shengmai Oral Liquid (S10) according to the method described in section "2.2.2". The solutions were injected separately according to the chromatographic conditions described in section "2.1". With psoralen (peak 9) as the reference peak, the RSD of the relative retention time of each common peak was calculated to be 0.02% to 0.27%, and the RSD of the relative peak area was 0.06% to 2.88%, indicating that the method has good repeatability.

[0056] 2.3.4 Fingerprint pattern establishment and similarity analysis

[0057] Following the chromatographic conditions described in section "2.1", 10 batches of Shenxian Shengmai oral liquid test solutions were analyzed. The data files were converted to "AIA" format and imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Using S10 as the reference chromatogram, with a time window width of 0.3, multi-point correction and Mark peak matching were performed, identifying 28 common peaks. A reference fingerprint chromatogram was generated using the median method. The overlay HPLC fingerprint chromatograms and reference chromatograms (R) of the 10 batches of Shenxian Shengmai oral liquid are shown in the appendix to the instruction manual. Figure 1 (A). The results showed that the similarity between the fingerprint spectra of the 10 batches of samples and the control was 0.934, 0.986, 0.994, 0.999, 0.987, 0.991, 0.994, 0.940, 0.995, and 0.991, respectively, all of which were greater than 0.93, indicating that the quality of different batches of Shenxian Shengmai oral liquid had good consistency.

[0058] 2.3.5 Identification of Common Peaks

[0059] Take the mixed reference solution (I) and the test solution separately, inject them according to the chromatographic conditions under section "2.1", and record the chromatograms. The results are as follows: Figure 1As shown, based on the retention time information of each chromatographic peak, 12 chromatographic peaks were identified as follows: peak 2 (tanshinone), peak 5 (pseudoephedrine hydrochloride), peak 9 (psoralen), peak 10 (isopsoralen), peak 11 (ferulic acid), peak 17 (rosmarinic acid), peak 20 (ginsenoside Rg1), peak 21 (tanshinone B), peak 24 (psoralen), peak 25 (isopsoralen), peak 27 (asparagine C), and peak 28 (icariin). Among them, peak 9 (psoralen) showed good separation, stable retention time, and a large peak area, and was therefore selected as the reference peak.

[0060] 2.4 Quantitative Analysis of Multiple Components in Shenxian Shengmai Oral Liquid

[0061] 2.4.1 Specificity test

[0062] Take blank methanol reference solution, mixed reference solution II, and test solution S10 respectively, and inject them into the chromatographic conditions described in section "2.1" for analysis. Record the chromatograms. Results can be found in the appendix to the instruction manual. Figure 2 As shown, the blank control solution showed no chromatographic peaks at the same retention time positions as the mixed standard solution, indicating that there was no interference with the analytes and the method had good specificity.

[0063] Examining linear relationships

[0064] Accurately pipette the mixed reference solution (II) and serially dilute it with methanol to six different suitable concentrations to obtain a series of mixed reference solutions. Analyze the solutions under the chromatographic conditions described in section "2.1" and determine the peak area of ​​each chromatographic peak. Plot the mass concentration of the indicator component on the x-axis (…). X The peak area is the vertical axis ( Y A standard curve was constructed, and the regression equation and correlation coefficient were calculated. The linear relationship of the 10 index components is shown in Table 1.

[0065] Table 1. Linear regression analysis of the 10 components

[0066]

[0067] 2.4.3 Precision Test

[0068] The same mixed reference solution was injected six times consecutively under the chromatographic conditions described in section “2.1”. The peak areas were measured, and the RSDs of the peak areas of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, rosmarinic acid, salvianolic acid B, psoralen, isopsoralen, ascorbic acid C, and icariin were calculated to be 1.95%, 0.82%, 1.80%, 1.80%, 1.82%, 1.68%, 1.89%, 1.84%, 1.42%, and 1.75%, respectively, indicating that the instrument precision was good.

[0069] 2.4.4 Stability Test

[0070] Take the same sample solution (S10) and, under the chromatographic conditions described in section “2.1”, inject and determine the peak area after standing at room temperature for 0, 2, 4, 8, 12 and 24 h, respectively. The RSDs of the peak areas of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, rosmarinic acid, salvianolic acid B, psoralen, isopsoralen, ascorbic acid C and icariin were calculated to be 1.98%, 1.31%, 1.88%, 1.83%, 1.73%, 0.86%, 2.48%, 2.69%, 1.70% and 1.82%, respectively, indicating that the sample is stable within 24 h at room temperature.

[0071] 2.4.5 Repeatability Test

[0072] Six test solutions were prepared in parallel using the same batch of Shenxian Shengmai Oral Liquid (S10) according to the method described in section "2.2.2". The solutions were analyzed under the chromatographic conditions described in section "2.1". The average contents of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, rosmarinic acid, salvianolic acid B, psoralen, isopsoralen, chomopodophyllin C, and icariin were calculated to be 1.864, 0.375, 3.900, 3.106, 0.166, 0.585, 0.245, 0.198, 0.420, and 0.467 mg·mL, respectively. -1 The RSDs were 1.71%, 2.90%, 1.62%, 1.63%, 1.74%, 1.59%, 2.73%, 2.77%, 1.41%, and 2.46%, respectively, indicating that the method has good repeatability.

[0073] 2.4.6 Recovery Test

[0074] Six portions (S10) of Shenxian Shengmai Oral Liquid with known content of the analyte were precisely measured. Ten reference standards for the analyte compounds were added sequentially at the 100% level to each portion. Sample solutions were prepared according to the method described in section "2.2.2" and analyzed under the chromatographic conditions described in section "2.1". Peak areas were measured, and the average recoveries of the ten components were calculated to be 99.0%–102.8%, with RSDs all less than 3%, indicating good method accuracy. Specific results are shown in Table 2.

[0075] Table 2 Recovery rates of 10 components (n = 6)

[0076]

[0077] 2.4.7 Sample Determination

[0078] Accurately pipette 0.1 mL from each of 10 batches of Shenxian Shengmai oral liquid, prepare sample solutions according to the method in section "2.2.2", and analyze them under the chromatographic conditions in section "2.1". Measure the peak area and calculate the content of 10 target components based on the linear regression equation. The specific measurement results are shown in Table 3.

[0079] Table 3. Determination of the content of 10 components in 10 batches of Shenxian Shengmai oral liquid (mg·mL) -1 (n=3)

[0080]

[0081] From Table 3 above, the content of tanshinone in 10 batches of samples was determined to be 1.218–2.781 mg·mL. -1 Pseudoephedrine hydrochloride 0.333–0.702 mg / mL -1 Psoralen 3.043–4.120 mg / mL -1 Isopsoralen 2.083–3.486 mg / mL -1 Rosmarinic acid 0.171–0.203 mg / mL -1 Danshensu B 0.158~1.046 mg·mL -1 Psoralen 0.189–0.286 mg / mL -1 Isopsoralen 0.196–0.323 mg / mL -1 , 0.231~0.403mg·mL of hormosiderin C -1 Icariin 0.323–0.512 mg / mL -1 The results of the upper and lower limits of the content detection of 10 effective substance indicators in the above 10 batches of Shenxian Shengmai oral liquid show that the content of the above effective indicator components has small changes, which can more comprehensively ensure the quality stability of Shenxian Shengmai oral liquid and ensure its clinical efficacy advantage.

[0082] Finally, it should be noted that this invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Any changes, equivalent substitutions, and improvements made by those skilled in the art under the guidance of this specification, within the spirit and scope of this invention, are within the protection scope of this invention.

Claims

1. A method for detecting multiple components of Shenxian Shengmai oral liquid, characterized in that, The detection method includes the following steps: (1) Mixed reference solution: Weigh out the reference standards of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, ferulic acid, rosmarinic acid, ginsenoside Rg1, salvianolic acid B, psoralen, isopsoralen, cuscuta citrate C and icariin, dissolve and dilute with methanol, mix well, and prepare a mixed reference solution. (2) Test solution: Take a sample of Shenxian Shengmai oral liquid, add methanol to dissolve and dilute it, filter it through a microporous membrane, and take the filtrate to obtain the test solution; (3) Chromatographic conditions: The chromatographic column is C10. 18 The mobile phase consisted of acetonitrile A and 0.2% phosphoric acid aqueous solution B, with gradient elution: 0 ~ 15 min, 5% A; 15 ~ 25 min, 5% ~ 15% A; 25 ~ 40 min, 15% ~ 18% A; For the time intervals of 40–45 min, 18%–22% A; 45–95 min, 22%–25% A; and 95–100 min, 25%–5% A, measurements were performed at 210 nm and 270 nm wavelengths, respectively. The detection wavelengths at 270 nm corresponded to 0–16 min and 24–38 min, while those at 210 nm corresponded to 16–24 min and 38–100 min. The flow rate was 0.6–1.4 mL / min. -1 The column temperature is 25–35°C, and the injection volume is 5–15 µL.

1. Chromatographic peak determination: Inject the mixed reference solution and test solution prepared using steps (1) and (2) above into the liquid chromatograph, and perform the determination according to the above steps. Under the specified chromatographic conditions, the chromatogram is determined and recorded to obtain the final result.

2. The detection method as described in claim 1, characterized in that, In step (1), the linear range of the contents of tanshinone, pseudoephedrine hydrochloride, psoralen, isopsoralen, rosmarinic acid, salvianolic acid B, psoralen, isopsoralen, chomopodophyllin C, and icariin in the mixed reference solution is 32.90~246.67 μg•mL, respectively. -1 9.60~71.67μg•mL -1 57.30~429.33μg•mL -1 54.50~408.00μg•mL -1 2.70~20.40 μg•mL -1 14.70~110.00μg•mL -1 7.00~52.67μg•mL -1 6.50 ~48.33 μg•mL -1 5.20 ~38.67 μg•mL -1 10.70~80.00 μg•mL -1 .

3. The detection method as described in claim 1, characterized in that, The steps The methanol in the test solution is diluted by a factor of 8 to 20.

4. The detection method as described in claim 3, characterized in that, The steps The methanol in the test solution was diluted 16 times.

5. The detection method as described in claim 1, characterized in that, The chromatographic conditions for step (3) are as follows: the column type is Ultimate LP.

6. The detection method as described in claim 1, characterized in that, The chromatographic conditions for step (3) are as follows: the detection flow rate is 1 mL·min. -1 The column temperature was 30°C and the injection volume was 10µL.

7. The detection method as described in claim 1, characterized in that, The steps Chromatographic peak determination was performed on 10 batches of test sample solutions. The detection data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The results showed that the similarity between the test sample solution and the control fingerprint spectrum was ≥0.93.

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