Fingerprint spectrum of musk-chuan NaoLiantong granules and detection method for quantitative analysis of multiple components by single marker

Abstract

The invention relates to and provides a fingerprint spectrum of musk-chuan NaoLiantong granules and a detection method for quantitative analysis of multiple components by single marker. The fingerprint spectrum detection method has the characteristics of simplicity and convenience in operation and good stability and reproducibility. 13 common peaks exist in an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of 15 batches of musk-chuan naoeutong granules, and 7 components in the fingerprint spectrum, namely geniposidic acid, chlorogenic acid, cowherb seed flavonoid glycoside, ferulic acid, senkyunolide I, salvianolic acid B and tanshinone IIA, are identified. The similarity of the 15 batches of the musk and chuan naoliantong granules is 0.961-1. According to the fingerprint spectrum detection method, 13 representative common characteristic peaks are determined so as to comprehensively reflect the internal quality characteristics of the musk-chuan naoliantong preparation.

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 fingerprint spectrum of Shechuan Naolitong Granules and its multi-evaluation detection method. Background Technology

[0002] Musk River Brain-Clearing Granules are a medical preparation developed by Xi'an Traditional Chinese Medicine Brain Disease Hospital to address the "blood stasis obstructing the brain orifices" pathogenesis of purulent meningitis. The medicine is composed of eight herbs: Chuanxiong (Ligusticum striatum), Danshen (Salvia miltiorrhiza), scorpion, artificial musk, leech, stir-fried Wangbuliuxing (Vaccaria segetalis), Plantago asiatica, and Lulutong (Liquidambar formosana). It is primarily used to treat obstructive hydrocephalus and purulent meningitis diagnosed as having the "blood stasis obstructing the brain orifices" pattern according to Traditional Chinese Medicine. Clinical studies have shown that this preparation can effectively improve consciousness disturbances in patients with purulent meningitis, significantly reduce the release of inflammatory mediators in the central nervous system and peripheral blood, and has good safety.

[0003] Fingerprint spectroscopy, as an important means of modern TCM quality control, has advantages such as comprehensiveness, systematicity, and reproducibility. This technology uses modern analytical methods such as high-performance liquid chromatography (HPLC) to obtain chromatographic peak information of multiple components in TCM, constructing a "fingerprint spectrum" that reflects its intrinsic chemical characteristics, thus enabling overall quality evaluation of TCM materials or preparations. Fingerprint spectroscopy is widely used in the stability studies, batch-to-batch consistency analysis, and adulteration identification of TCM, and is one of the key technologies in the modernization of TCM. However, traditional fingerprint spectroscopy evaluation indicators are mostly limited to peak shape and similarity analysis, lacking quantitative control of key pharmacodynamic components, making it difficult to meet the quality evaluation requirements of "quality-efficacy consistency" in modern TCM preparations. Therefore, multi-component analysis has emerged as an effective supplementary means for TCM content determination. This method, based on the similarity in the structural and physicochemical properties of components in TCM, selects a stable and readily available component as an internal reference. By measuring its content and combining it with a relative correction factor, the content of multiple target components in the system can be simultaneously calculated, thereby achieving simultaneous evaluation of multiple components. The one-test-multiple-evaluation technique can effectively save reference standard resources and reduce testing costs, and is especially suitable for research on traditional Chinese medicine compound preparations where reference standards are scarce or expensive. Organically combining traditional Chinese medicine fingerprinting with the one-test-multiple-evaluation method can take into account both the overall chemical information of the preparation and the quantitative control of key components, breaking through the limitations of traditional Chinese medicine quality control that relies on "single components" and "single standards." It represents the modernization and scientific development direction of traditional Chinese medicine quality control and has been applied in many national drug standards and new drug research and development practices.

[0004] Currently, the quality control of Shechuan Naolitong granules mainly relies on thin-layer chromatography for identification, and there are no publicly available studies or reports on the establishment of its fingerprint spectrum and the determination of its effective component content. Therefore, there is an urgent need to establish a scientific and systematic quality control method to achieve standardized and regulated management of its formulation. Summary of the Invention

[0005] This invention relates to a fingerprint spectrum of Shechuan Naolitong granules and a multi-evaluation detection method thereof. This fingerprint spectrum detection method is characterized by its simplicity, stability, and good reproducibility. Furthermore, by identifying 13 common peaks in the HPLC fingerprint spectra of 15 batches of Shechuan Naolitong granules, seven components in the fingerprint spectrum were identified: genipinic acid (peak 3), chlorogenic acid (peak 6), Wangbuliuxing flavonoid glycosides (peak 8), ferulic acid (peak 9), chuanxiong lactone I (peak 11), tanshinone B (peak 12), and tanshinone IIA (peak 13). The similarity among the 15 batches of Shechuan Naolitong granules ranged from 0.961 to 1. The 13 representative common characteristic peaks identified by the fingerprint spectrum detection method of this invention comprehensively reflect the intrinsic quality characteristics of Shechuan Naolitong preparations.

[0006] This invention also establishes a multi-dimensional content detection method to determine the content of six main components: genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, ligustrazine lactone I, and salvianolic acid B. This method is highly accurate, reproducible, consumes few reference standards, and can simultaneously assess overall chemical characteristics and quantitatively analyze key components. It is suitable for quality evaluation during the production and regulatory processes of Shechuan Naolitong granules. These indicative components may be related to the material basis of Shechuan Naolitong granules in inhibiting inflammation, protecting the nervous system, and improving microcirculation. This method can be used as the detection method for the internal control quality standard of the granules in this invention in large-scale industrial production.

[0007] The technical solution of this invention patent application is as follows:

[0008] A fingerprint detection method for Shechuan Naolitong granules, the detection method comprising the following steps:

[0009] (1) Preparation of test solution: Take the powder of musk naolitong granules, add 65-75% methanol, seal tightly, weigh, sonicate, cool, make up the loss, shake well, filter, evaporate to dryness, dissolve the residue in 65-75% methanol, make up to volume, shake well, filter, and take the filtrate to obtain the test solution.

[0010] (2) Preparation of mixed reference solution: Accurately weigh appropriate amounts of geniposide, chlorogenic acid, limonene flavonoids, ferulic acid, ligustrazine lactone, and salvianolic acid B reference standards, and dissolve them in 70% methanol to prepare a mixed reference solution. The concentration of geniposide is 150–200 μg / mL, the concentration of chlorogenic acid is 150–170 μg / mL, the concentration of limonene flavonoids is 70–90 μg / mL, the concentration of ferulic acid is 130–170 μg / mL, the concentration of ligustrazine lactone is 160–190 μg / mL, and the concentration of salvianolic acid B is 700–740 μg / mL. Mix thoroughly to obtain the solution.

[0011] (3) Chromatographic conditions: C18 column; mobile phase: acetonitrile (phase A); 0.1% phosphoric acid aqueous solution (phase B); gradient elution ratios: 0–7 min, mobile phase A 5%, mobile phase B 95%; 7–12 min, mobile phase A 5%–10%, mobile phase B 95%–90%; 12–40 min, mobile phase A 10%–22%, mobile phase B 90%–78%; 40–60 min, mobile phase A 22%–25%, mobile phase B 78%–75%; 60–80 min, mobile phase A 25%–45%, mobile phase B 75%–55%; 80–85 min, mobile phase A 45%–70%, mobile phase B 55%–30%; 85–95 min, mobile phase A 70%–90%, mobile phase B 30%–10%; 95–105 min, ... At 105–110 min, mobile phase A is 90% and mobile phase B is 10%; at 105–110 min, mobile phase A is 90%–5% and mobile phase B is 10%–95%.

[0012] 110–120 min, mobile phase A is 5%, mobile phase B is 95%; detection wavelength is 260–300 nm, column temperature is 25–32℃;

[0013] (4) Establish fingerprint chromatogram: Take the test solution from step (1) and the mixed reference solution from step (2), and determine them using the chromatographic conditions in step (3). Perform the detection according to the chromatographic conditions in step (3), and analyze multiple batches of the test solution of Shechuan Naolitong Granules. Import the samples into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to generate a reference fingerprint chromatogram.

[0014] Preferably, in step (1) of the detection method, the ultrasonic frequency is 35~45kHz, the ultrasonic power is 350~450W, and the ultrasonic processing time is 20~40min.

[0015] Preferably, the ultrasonic frequency is 40Hz, the ultrasonic power is 400W, and the ultrasonic processing time is 30min.

[0016] Preferably, the concentration of methanol in step (2) of the detection method is 70%.

[0017] Preferably, in step (2) of the detection method, the concentration of the mixed reference solution is 184.0 μg / mL for genipinic acid, 172.0 μg / mL for chlorogenic acid, 78.2 μg / mL for vaccaria segetalis flavonoid glycoside, 153.2 μg / mL for ferulic acid, 178.4 μg / mL for ligusticum lactone, and 722.0 μg / mL for salvianolic acid B.

[0018] Preferably, in step (3) of the detection method, the chromatographic column C... 18The model number is Kromasil 100-5, and the column specifications are 250 mm × 4.6 mm and 5 μm.

[0019] Preferably, in step (3) of the detection method, the volumetric flow rate is 1.0 mL / min, the detection wavelength is 254 nm and 280 nm, and the column temperature is 25 °C.

[0020] Preferably, the test solution is detected according to the chromatographic conditions in step (3), and the generated liquid chromatographic peak is compared with the reference fingerprint spectrum, with a similarity of 0.90 to 1.00.

[0021] Preferably, the detection method is a multi-evaluation content detection method, including the following steps: the preparation of the reference standard and the test solution and the chromatographic conditions are the same as steps (1) to (3) of claim 1, and the contents of genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycoside, ferulic acid and chuanxiong lactone I are determined using tanshinone B as the reference peak.

[0022] Preferably, the chromatographic conditions for determining the content of genipin, chlorogenic acid, vacuolated flavonoids, ferulic acid, ligustrazine lactone I, and salvianolic acid B are as follows: flow rate 1.0 mL / min, column temperature 30℃, injection volume 10 μL, wherein the detection wavelength of genipin is 254 nm, and the detection wavelengths of chlorogenic acid, vacuolated flavonoids, ferulic acid, ligustrazine lactone I, and salvianolic acid B are 280 nm.

[0023] Preferably, the detection method is used for the quality detection of Shechuan Naolitong granules, and for the identification of intermediates and effective components of traditional Chinese medicine.

[0024] To further illustrate the inventiveness of the detection method for Shechuan Naolitong granules of the present invention, some of the experimental contents of the screening of the technical solution of the present invention are summarized as follows:

[0025] Because the granules of Muscone-containing Naolitong contain a wide variety of components, and the polarity of the effective components in the traditional Chinese medicine compound varies greatly, isocratic elution is difficult to achieve effective separation of the target components and can easily cause peak overlap and tailing, affecting the accuracy of qualitative and quantitative analysis. Therefore, this invention employs gradient elution. Through systematic exploration and repeated experiments, the mobile phase combination and gradient program are gradually optimized to improve peak shape quality and resolution, achieving the goal of simultaneous separation and detection of multiple components.

[0026] Table 1. Partial Experimental Exploration of Gradient Elution of Musk River Brain-Clearing Particles in Mobile Phase

[0027]

[0028] In the initial condition exploration, this invention attempted to use acetonitrile (A) – 0.1% phosphoric acid aqueous solution (B) as the mobile phase, comparing the separation effects among multiple gradient schemes. Experiments revealed that if the acetonitrile ratio increased too rapidly in the first 20 minutes, it caused a significant baseline shift, affecting the resolution of subsequent chromatographic peaks; while if the ratio increased too slowly, it caused early component tailing and peak broadening. Ultimately, by adjusting the acetonitrile infusion rate, the analysis time was controlled within a reasonable range while ensuring system stability. Specifically, in the 0–7 min stage, 5% acetonitrile was maintained to achieve initial separation of early peak elution. From 7–12 min, acetonitrile was slowly increased to 10%, effectively shortening the elution time of genipinic acid and chlorogenic acid. From 12–40 min, acetonitrile increased from 10% to 22%, providing a sufficient separation window for polar components such as chlorogenic acid and Wangbuliuxing flavonoid glycosides. From 40–60 min, the acetonitrile was further increased to 25%, successfully separating moderately polar compounds such as ferulic acid and ligustrazine lactone I. From 60–85 min, acetonitrile increased from 25% to 70%, gradually eluting high-retention-time components such as salvianolic acid B and avoiding peak congestion in the later stages. Among these, genipinic acid and chlorogenic acid, and Wangbuliuxing flavonoid glycosides and ferulic acid, had similar retention times, posing separation challenges. This invention solves the problems of peak overlap and baseline drift by adjusting the gradient slope and changing the wavelength through multiple rounds of experiments. The separation degree of all components is greater than 1.5, the retention time is stable, and the accuracy of subsequent content determination is ensured.

[0029] Furthermore, to improve detection efficiency, reduce solvent consumption, and minimize environmental pollution, this invention does not employ an excessive organic solvent rinsing strategy. Instead, it significantly shortens the total analysis time to less than 120 minutes while ensuring separation effectiveness through a rationally designed elution program. Addressing the dual objectives of separation effectiveness and detection cost, this invention, after thoroughly validating different gradient schemes, ultimately established the following optimal gradient elution conditions: 0–7 min, 5% acetonitrile (A); 7–12 min, 5%–10% A; 12–40 min, 10%–22% A; 40–60 min, 22%–25% A; 60–80 min, 25%–45% A; 80–85 min, 45%–70% A; 85–95 min, 70%–90% A; 95–105 min, maintain 90% A; 105–110 min, 90%–5% A; 110–120 min, maintain 5% A to rebalance the system.

[0030] Compared to existing methods that primarily employ single-component quantitative analysis or single-wavelength fingerprinting for quality control, this invention offers significant advantages in the following aspects: ① **Multi-indicator integration enhances the comprehensiveness of quality control:** Current methods mostly monitor only 1-2 components or establish non-quantitative fingerprints, failing to comprehensively characterize drug quality. This invention simultaneously establishes fingerprints with 13 common peaks and performs qualitative and quantitative analysis of 6 key components, combining breadth and depth to enhance the scientific rigor of quality control. ② **One-test-multiple-evaluation technology reduces detection costs:** Conventional methods often use expensive and frequently used reference standards such as chlorogenic acid and Wangbuliuxing flavonoid glycosides. This invention employs QAMS technology, using Danshensu B as an internal control to replace multiple reference standards, achieving "one injection, multiple component determination," effectively saving experimental costs. ③ **Significantly improved chromatographic resolution and sensitivity:** Current methods suffer from issues such as partial peak overlap and quantification difficulties. This invention, through optimized gradient design, achieves resolutions greater than 1.5, good peak shapes, and demonstrates high sensitivity and repeatability, making it suitable for complex matrix samples. ③ The method has wide applicability and is easy to promote and apply: This method is applicable to multiple stages such as laboratory research, production quality control, and market supervision. It has good repeatability, stability and scalability, and can serve as an important technical support for the quality standard of Shechuan Naolitong Granules.

[0031] In summary, this invention overcomes the technical bottlenecks of difficult separation, difficult quantification, and high cost in the process of screening and optimizing chromatographic separation conditions, demonstrating significant technical innovation and wide application value, and showing a clear improvement and inventiveness over existing technologies.

[0032] The beneficial effects of the patented technical solution of this invention are as follows:

[0033] (1) This study established a method for determining the content of genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, and ligustrazine lactone I using fingerprinting combined with a single-analysis-multiple-evaluation approach. This method not only improves detection efficiency but also reduces analytical costs, providing an important reference for improving the quality standards of Shechuan Naolitong Granules. The optimal chromatographic conditions for the fingerprint detection method of this invention, obtained through extensive experimentation, are as follows: acetonitrile (A)-phosphoric acid aqueous solution (0.1%, B) as the mobile phase, with gradient elution (0–7 min, 5% A; 7–12 min, 5%–10% A; 12–40 min, 10–22%; 40–60 min, 22%–25% A; 60–80 min, 25–45% A; 80–85 min, 45%–70% A; 85–95 min, 70%–90% A; 95–105 min, 90% A; 105–110 min, 90%–5% A; 110–120 min, 5% A).

[0034] (2) A standard fingerprint chromatogram was generated according to the preferred detection method of this invention. Using the chromatogram of sample S11 as a reference chromatogram, a superimposed chromatogram and a control chromatogram (R) were generated using the median method and common peak matching. A total of 13 common peaks were identified. By comparing the retention time and UV absorption spectrum information with the reference standard, seven medicinal active ingredients were identified: genipinic acid (peak 3), chlorogenic acid (peak 6), Wangbuliuxing flavonoid glycosides (peak 8), ferulic acid (peak 9), chuanxiong lactone I (peak 11), tanshinone B (peak 12), and tanshinone IIA (peak 13). Furthermore, similarity calculations were performed, and the similarity between the 15 batches of samples and the control chromatogram was in the range of 0.961 to 1. Modern pharmacological experiments show that genipinic acid can reduce the inflammatory cascade reaction caused by meningitis, reduce free radical damage, and protect brain tissue. Chlorogenic acid can inhibit Streptococcus pneumoniae and Staphylococcus aureus, reducing bacterial spread in cerebrospinal fluid and suppressing meningitis inflammatory storms; it also reduces brain nerve damage through antioxidant (reducing ROS) and anti-apoptotic (regulating Bcl-2 / Bax) effects. Wangbuliuxing flavonoids can inhibit neuroinflammation, enhance blood-brain barrier repair, improve microcirculation, and reduce cerebral edema by promoting vascular endothelial growth factor expression. Ferulic acid reduces oxidative stress damage and cerebral edema by activating the Nrf2 / HO-1 pathway; it also inhibits Caspase-3-mediated neuronal apoptosis, reducing the likelihood of sequelae cognitive impairment. Ligusticum striatum lactone I reduces the release of pro-inflammatory cytokines, inhibits neuroinflammation, and simultaneously enhances cerebral microcirculation and reduces cerebral edema by inhibiting the MAPK / NF-κB signaling pathway and the transcription and expression of microglia. Tanshinone B significantly improves the activation of microglia in the brain tissue of young mice with pneumonia, inhibits the inflammatory response in brain tissue, and thus exerts a neuroprotective effect. Furthermore, the pharmacological activities of the effective components identified in this invention are all related to the indications and functions of the traditional Chinese medicine granules of this invention. This study identified and detected the contents of the above-mentioned components through fingerprinting, suggesting that genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, chuanxiong lactone I, and tanshinone B may be the material basis for the anti-inflammatory, nervous system-protective, and microcirculation-improving effects of Shechuan Naolitong granules. In addition, tanshinone IIA, as the main active substance for the efficacy of tanshinone, has been proven to protect against hippocampal damage in a rat model of bacterial meningitis by inhibiting the NF-κB pathway.

[0035] (3) Fingerprint similarity analysis of the present invention: The similarity of the 15 batches of Shechuan Naolitong granules test solutions were 0.961, 0.999, 0.999, 1.000, 1.000, 1.000, 0.999, 0.997, 0.994, 0.988, 1.000, 0.998, 0.999, 0.999, and 0.999, respectively. This indicates that the similarity between the 15 batches of samples and the control chromatogram is within the range of 0.961 to 1. This also shows that the quality of different batches of Shechuan Naolitong granules in this invention is uniform and the stability is good.

[0036] (3) Methodological investigation of the fingerprint detection of this invention: Precision test results: The relative standard deviation (RSD) of the relative retention time of each common peak is ≤0.3%, and the relative peak area RSD is ≤0.8%, indicating good precision. Repeatability test results: The calculated relative retention time RSD of each common peak is ≤0.7%, and the relative peak area RSD is ≤1.6%, indicating good repeatability of the method. Stability test results: The calculated relative retention time RSD of each common peak is ≤0.4%, and the relative peak area RSD is ≤0.9%, indicating good stability of the test solution within 24 h.

[0037] (4) This invention also establishes a method for determining the content of six main components—genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, and ligustrazine I—using a dual-wavelength method at 254 nm and 280 nm as the internal reference peak and a correction factor method. This method is highly accurate, reproducible, consumes few reference standards, and can simultaneously assess overall chemical characteristics and quantitatively analyze key components. It is suitable for quality evaluation of Shechuan Naolitong granules during production and regulatory processes.

[0038] Instrument precision test results: The RSD of each chromatographic peak was less than 2.1%, indicating good instrument precision. Repeatability test results: The RSD of each chromatographic peak was less than 3.3%, indicating good method repeatability. Stability test results: The RSD of each chromatographic peak was less than 1.7%. Spiking recovery test results: The recoveries of genipinic acid, genipinic acid, Wangbuliuxing flavonoid glycoside, ferulic acid, chuanxiong lactone I, and tanshinone B were 93.15%, 94.06%, 99.09%, 93.71%, 99.46%, and 100.46%, respectively, with RSDs of 2.4%, 2.1%, 0.5%, 1.5%, 1.2%, and 1.0%, respectively, indicating good accuracy of the analytical method.

[0039] In summary, this invention establishes a method for determining the content of genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, and ligustrazine lactone I using fingerprinting combined with a single-method-multiple-measurement approach. This method not only improves detection efficiency but also reduces analytical costs. Methodological evaluation shows that the established method is highly specific, reproducible, accurate, and reliable, providing an important reference for improving the quality standards of Shechuan Naolitong Granules. Attached Figure Description

[0040] Figure 1 HPLC fingerprints of 15 batches of Shechuan Naolitong granules;

[0041] Figure 2Chromatograms of blank solvent (A), mixed reference standard (B), and muskwort granules (C), including 3-genipin glycoside; 6-chlorogenic acid; 8-wolfowl flavonoid glycoside; 9-ferulic acid; 11-ligustrazine lactone I; 12-tanshinone B; 13-tanshinone IIA;

[0042] Figure 3 OPLS-DA score plots of different batches of Muscone-containing brain-tonifying samples;

[0043] Figure 4 VIP images of different batches of Muscone-containing brain-tonifying samples. Detailed Implementation

[0044] 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, wherein GA - genipinic acid, CA - chlorogenic acid, VA - wangbuliuxing flavonoid glycoside, FA - ferulic acid, SI - chuanxiong lactone I, SAB - tanshinone B, TA - tanshinone IIA, ESM - external standard method, QAMS - one test with multiple evaluations method.

[0045] Example 1: Construction of the fingerprint spectrum of *Musk River Brain Litong* according to the present invention

[0046] 1. Instruments and Materials

[0047] 1.1 Instruments

[0048] Agilent 1260 HPLC system (Agilent Technologies, USA); Shimadzu LC-2010 HT HPLC system (Shimadzu Laboratory Instruments, Japan); SQP 0.0001 ppm electronic analytical balance (Sartorius Scientific Instruments, Ltd.); ME55 0.0001 ppm electronic analytical balance (Mettler Toledo, Switzerland); Kromasil 100-5-C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm, Nouryon AG, Sweden); Agilent Zorbax SB-Aq C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, USA); Welch Ultimate LP-C 18 Chromatographic column (250mm×4.6mm, 5μm, Shanghai Yuexu Technology Co., Ltd.); XM-P22H stepless adjustable ultrasonic cleaner (Xiaomei Ultrasonic Instrument Co., Ltd.).

[0049] 1.2 Materials

[0050] Musk River Brain-Clearing Granules (Pharmaceutical Registration No.: Shaanxi Pharmaceutical Preparation No. Z20230408000, Batch Nos. 20220701, 20220703, 20220901, 20221001, 20230201, 20230202, 20230501, 20230503, 20230701, 20230702, 20231001, 20231101, 20240101, 20240203, 20241101, S1~S15 in sequence), Xi'an Traditional Chinese Medicine Brain Disease Hospital, Specification: 5 g per bag); Acetonitrile was chromatographic grade, water was Wahaha purified water, and other reagents were analytical grade; Reference standard geniposidic acid (Batch No. O30IB228770, geniposidic acid) The following acids were purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a mass fraction ≥98% (GA), chlorogenic acid (A22GB158496, CA), vaccarin (G24J11L119380, VA), ferulic acid (JB255905, FA), senkyunolide I (M21HB178706, SI), salvianolic acid B (JB298883, SAB), and tanshinone IIA (JB278528, TA).

[0051] 1.3 Methods and Results

[0052] 1.3.1 Chromatographic conditions

[0053] Kromasil 100-5-C 18 The chromatographic column (250 mm × 4.6 mm, 5 μm) was used with acetonitrile (A)-phosphoric acid aqueous solution (0.1%, B) as the mobile phase, and gradient elution was performed (0–7 min, 5% A; 7–12 min, 5%–10% A; 12–40 min, 10–22%; 40–60 min, 22%–25% A; 60–80 min, 25–45% A; 80–85 min, 45%–70% A; 85–95 min, 70%–90% A; 95–105 min, 90% A; 105–110 min, 90%–5% A; 110–120 min, 5% A). The flow rate was 1.0 mL / min, the detection wavelengths were 254 and 280 nm, the column temperature was 25 ℃, and the injection volume was 5 μL.

[0054] 1.3.2 Solution Preparation

[0055] 1.3.2.1 Preparation of mixed reference solution

[0056] Take appropriate amounts of GA (genipinic acid), CA (chlorogenic acid), VA (vacoside), FA (ferulic acid), SI (ligustrazine lactone I), and SAB (tanshinone B) reference standards, accurately weigh them, and dissolve them in 70% methanol to prepare a mixed reference standard solution containing GA 184.0 μg / mL, CA 172.0 μg / mL, VA 78.2 μg / mL, FA 153.2 μg / mL, SI 178.4 μg / mL, and SAB 722.0 μg / mL. Mix thoroughly to obtain the final solution.

[0057] 1.3.2.2 Preparation of the test solution

[0058] Accurately weigh approximately 0.5 g of malachite granules (passed through a No. 4 sieve), place them in a stoppered conical flask, accurately add 25 mL of 70% methanol, seal tightly, weigh, sonicate for 30 min, cool, make up the volume loss, shake well, filter, evaporate to dryness, dissolve the residue in 70% methanol and dilute to 5 mL in a volumetric flask, shake well, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain the final product.

[0059] 1.4 Fingerprint Analysis

[0060] 1.4.1 Specificity

[0061] The above-mentioned mixed reference solution, test solution, and blank solvent (70% methanol) were injected under the chromatographic conditions described in section "1.3.1". The results showed that the blank solvent did not absorb at the peak positions of any reference component, indicating that the solvent had no effect on the chromatographic peaks. The chromatographic peaks of each reference component in both the mixed reference solution and the test solution showed good resolution, demonstrating the good specificity of this method. The chromatographic results are shown below. Figure 1 .

[0062] 1.4.2 Instrument Precision

[0063] The same batch of Shechuan Naolitong granules sample test solution (No.: S11) was continuously injected and measured 6 times under the chromatographic conditions in section "1.3.1". With peak No. 12 as the reference peak (S peak), the relative standard deviation (RSD) of the relative retention time of each common peak was calculated to be ≤1.61%, and the relative peak area RSD was ≤2.82%, indicating that the instrument precision is good.

[0064] Table 2 Calculation results of relative retention time for fingerprint precision

[0065]

[0066] Table 3. Calculation results of relative peak area for fingerprint precision

[0067] The results show that the RSD of the relative retention time of the common peak is less than 0.3%, and the RSD of the relative peak area is less than 0.8%, indicating that the instrument has good precision.

[0068] 1.4.3 Repeatability:

[0069] Six aliquots of the test solution were prepared in parallel according to the preparation method of the test solution, and each aliquot was injected for analysis. The retention time and peak area of ​​the chromatographic peaks were determined. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated with peak 12 (tanshinone B) as the reference peak to examine the repeatability of the method. The results are shown in Tables 4 and 5.

[0070] Table 4. Calculation results of relative retention time for fingerprint repeatability.

[0071]

[0072] Table 5. Calculation results of relative peak area for fingerprint repeatability.

[0073]

[0074] The results show that the RSD of the relative retention time of the common peak is less than 0.7%, and the RSD of the relative peak area is less than 1.6%, indicating good reproducibility of the method.

[0075] 1.4.4 Stability

[0076] The test solution was injected and analyzed at 0, 2, 4, 8, 12, and 24 hours to determine the retention time and peak area of ​​the chromatographic peaks. Peak 12 (tanshinone B) was used as the reference peak to calculate the RSD values ​​of the relative retention time and relative peak area of ​​each common peak. The stability of the test solution was investigated, and the results are shown in Tables 6 and 7.

[0077] Table 6. Calculation results of relative retention time for fingerprint spectrum stability.

[0078]

[0079] Table 7. Calculation results of relative peak area for fingerprint spectrum stability.

[0080]

[0081] The results show that the RSD of the relative retention time of the common peak is less than 0.4%, and the RSD of the relative peak area is less than 0.9%, indicating good reproducibility of the method.

[0082] 1.4.5 Establishment of Comparative Fingerprint Patterns

[0083] Fifteen batches of Shechuan Naolitong Granules were prepared according to the preparation method of Shechuan Naolitong Granules. Fifteen batches of test solutions were prepared according to the method determined above. The fingerprint chromatograms of each test solution were recorded by injecting them into a liquid chromatogram. The fingerprint chromatograms of the 15 batches were then imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to generate a control fingerprint chromatogram (see attached). Figure 2 The similarity between the fingerprint chromatograms of 15 batches of Shechuan Naolitong granules and the control was calculated based on common peaks. According to the similarity calculation of the fingerprint chromatograms, the similarity range between the fingerprint chromatograms of the 15 batches of Shechuan Naolitong granules and the control was 0.961~1.000, all with a similarity greater than 0.90. The fingerprint chromatogram of the test sample should show chromatographic peaks with the same retention time as the chromatographic peaks of the control, and the chromatogram of the test sample should be basically consistent with the fingerprint chromatogram of the control, with 13 corresponding common peaks. According to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity calculated based on common peaks should not be less than 0.90 between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the control.

[0084] 1.4.6 Peak identification and assignment based on fingerprint spectrum

[0085] Following the fingerprint chromatographic determination method, the test solution of Shechuan Naolitong granules, Danshensu B, Genipin glycoside, Chlorogenic acid, Wangbuliuxing flavonoid glycoside, Ferulic acid, and Ligusticum striatum lactone I reference solution were injected separately, and the chromatograms were recorded to determine the assignment of each chromatographic peak in Shechuan Naolitong granules. The experimental results are shown in Table 8.

[0086] Table 8. Attribution of chromatographic peaks in fingerprint chromatograms.

[0087]

[0088] 1.5 Fingerprint pattern establishment and similarity evaluation

[0089] Fifteen batches of the test solution of Shechuan Naolitong Granules were injected and analyzed according to the chromatographic conditions in section "1.3.1". The chromatograms were recorded, and the chromatographic information was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012.130723)". Using the chromatogram of sample S11 as the reference chromatogram, the median method was used to generate a superimposed chromatogram and a reference chromatogram (R) through common peak matching. A total of 13 common peaks were identified. See [link to relevant documentation]. Figure 2By comparing the retention time and UV absorption spectrum information with the reference standard, seven components were identified: genipinic acid (peak 3), chlorogenic acid (peak 6), Wangbuliuxing flavonoid glycosides (peak 8), ferulic acid (peak 9), chuanxiong lactone I (peak 11), tanshinone B (peak 12), and tanshinone IIA (peak 13). Furthermore, similarity calculations were performed, and the results are shown in Table 9. The similarity between the 15 batches of SCNLT samples and the reference spectrum ranged from 0.961 to 1, and the RSD of the peak area of ​​the common peaks across batches ranged from 6.18% to 50.85%, indicating that different batches of the preparation have similar types of components overall, but differences in content.

[0090] Table 9. Similarity evaluation of 15 batches of Shechuan Naolitong samples.

[0091]

[0092] 1.6 Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) and Variable Importance Projection (VIP) Analysis of Common Peaks in Fingerprint Spectra

[0093] The peak areas of 13 common peaks from 15 batches of SCNLT samples were imported into SIMCA 14.1 software for OPLS-DA, and the results were displayed. 2 X, R 2 Y was 0.969 and 0.722 respectively, and the model's predictive effectiveness index Q was... 2 The value is 0.535. Samples prepared from similar batches of medicinal materials cluster together. Among them, S1 and S6-S10 are evenly distributed on the right side of the horizontal axis "0", while S2-S5 and S11-S15 are each clustered on the other side. See Figure 3 Furthermore, 200 permutation tests were performed on the established OPLS-DA model, and the intercept R was found to be... 2 =0.093, Q 2 =-0.456, and R 2 and Q 2 The positive slope of the regression line indicates that the model is not overfitting and can be used for further data analysis. VIP is an important indicator of inter-group differences; compounds with quality differences between different batches of drugs are usually screened using VIP > 1. Results are shown below. Figure 3 As can be seen from the figure, the VIP of peaks 12, 5 and 11 is greater than 1, which may be the potential differential components of the 15 batches of SCNLT samples. Among them, peaks 11 and 12 were identified by the reference standard as ligustrolactone I and salvianolic acid B, respectively.

[0094] Example 2: Validation of the method for determining the content of six components: genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, ligustrazine lactone I, and salvianolic acid B.

[0095] Chromatographic conditions: Kromasil 100-5-C18 The chromatographic column (250 mm length, 4.6 mm inner diameter, 5 μm particle size) was used. Gradient elution was performed using acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B) as the mobile phase. Genipin glycosides were detected at 254 nm, while chlorogenic acid, vaccaria segetalis flavonoids, ferulic acid, ligustrazine lactone I, and salvianolic acid B were detected at 280 nm. The column temperature was 30 °C, and the flow rate was 1.0 mL / min. The gradient elution ratio, preparation parameters for the reference solution, and preparation parameters for the test solution were the same as in Example 1.

[0096] 2.1 Exclusivity:

[0097] To investigate whether the blank solvent interfered, genipinic acid, chlorogenic acid, Wangbuliu flavonoid glycosides, ferulic acid, ligustrolactone I, and salvianolic acid B mixed reference solution, test solution, negative sample solution, excipient blank solution (dextrin-sucrose powder mixture), and blank solvent (70% methanol) were injected into the liquid chromatograph, and the chromatograms were recorded. The results showed that neither the blank solvent (70% methanol) nor the excipient blank (dextrin-sucrose powder mixture) interfered with the determination of genipinic acid, chlorogenic acid, Wangbuliu flavonoid glycosides, ferulic acid, ligustrolactone I, and salvianolic acid B, indicating that the method has good specificity and also meets the principle of maximizing information collection.

[0098] 2.2 Examination of linear relationships:

[0099] Accurately pipette 0.1, 0.5, 1.0, 1.5, and 2.0 mL of the above mixed reference solution, and dilute to 5 mL with 70% methanol. Shake well to obtain a series of mixed reference solutions of different concentrations. Inject each of these solutions for analysis. Plot a standard curve with the peak area of ​​GA reference at 254 nm and the peak areas of CA, VA, FA, SI, and SAB references at 280 nm as the ordinate (Y) and the corresponding concentration as the abscissa (X). The compounds showed good linearity within their respective ranges. The results are shown in Table 10.

[0100] Table 10 shows the linearity results of the six components.

[0101]

[0102] 2.3 Instrument Precision

[0103] A mixed standard solution containing genipin, chlorogenic acid, vasiloxane flavonoids, ferulic acid, ligustrazine lactone I, and salvianolic acid B was injected six times consecutively. The peak area results are shown in Table 11.

[0104] Table 11 Results of instrument precision test (n=6).

[0105]

[0106] The experimental results show that the peak area RSD of each reference standard is ≤2.1%, indicating that the instrument has good precision.

[0107] 2.4 Repeatability Test

[0108] The experimenter (A) took 6 portions of this product and prepared 6 test solutions according to the established method for preparing and determining the test solution. The RSD of the content of the 6 samples was calculated and the average value of the content of the 6 samples was taken as the content of this batch of samples. The results are shown in Table 12.

[0109] Table 12 Results of repeatability tests (n=6).

[0110]

[0111] The experimental results showed that the RSD of each component in the 6 samples was less than 3.3%, indicating that the method had good repeatability.

[0112] 2.5 Stability Test

[0113] Following the established method for preparing and determining the test solution, the test solution was injected and determined after being left to stand for 0, 2, 4, 8, 12, and 24 hours. The RSD of the sample content was calculated, and the results are shown in Table 13.

[0114] Table 13 Results of instrument precision test (n=6).

[0115]

[0116] Experimental results show that the RSD of each chromatographic peak area in the test sample is ≤1.7% within the first 24 hours, proving that the test sample solution of the present invention has good stability within 24 hours.

[0117] 2.6 Accuracy Test (Recovery Rate)

[0118] Using the spiking recovery method, appropriate amounts of genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, ligustrazine lactone I, and salvianolic acid B reference standards were accurately weighed and dissolved in 70% methanol to prepare a mixed reference solution containing genipinic acid 184.0 μg / mL, chlorogenic acid 172.0 μg / mL, Wangbuliuxing flavonoid glycosides 78.2 μg / mL, ferulic acid 153.2 μg / mL, SI 178.4 μg / mL, and SAB 722.0 μg / mL. In the repeatability experiment, the contents of geniposide were determined to be 0.1626 mg / g, chlorogenic acid 0.1150 mg / g, vaccaria segetalis flavonoid glycosides 0.1823 mg / g, ferulic acid 0.0968 mg / g, ligustrolactone I 0.1044 mg / g, and salvianolic acid B 1.8086 mg / g in S11. The results are shown in Tables 14-19.

[0119] Designed with a ratio of approximately 1:1 between the reference standard and the test sample, 0.25 g of S11 powder with known content was taken in 6 portions. Each portion was precisely mixed with 25 mL of 70% methanol containing 38.64 µg of genipin, 27.52 µg of chlorogenic acid CA, 44.574 µg of Wangbuliuxing flavonoid glycoside, 22.98 µg of ferulic acid, 24.976 µg of ligusticum lactone I, and 447.64 µg of salvianolic acid B to prepare the test solution and then injected for determination.

[0120] Table 14 Results of the accuracy test for genipin acid content determination (n=6).

[0121]

[0122] The experimental results showed that the average recovery rate was 93.15% and the RSD was <3.0%, indicating that the method has good accuracy in determining the content of genipinic acid.

[0123] Table 15 Results of the accuracy test for chlorogenic acid content determination (n=6).

[0124]

[0125] The experimental results showed that the average recovery rate was 94.06% and the RSD was <3.0%, indicating that the method has good accuracy in determining chlorogenic acid.

[0126] Table 16 Results of the accuracy test for the determination of flavonoid glycoside content in Vaccaria segetalis (n=6).

[0127]

[0128] The experimental results showed that the average recovery rate was 99.09% and the RSD was <0.5%, indicating that the method has good accuracy in determining the content of flavonoid glycosides in Vaccaria segetalis.

[0129] Table 17 Results of the accuracy test for ferulic acid content determination (n=6).

[0130]

[0131] The experimental results showed that the average recovery rate was 93.71% and the RSD was <1.5%, indicating that the method has good accuracy in determining the content of ferulic acid.

[0132] Table 18 Results of the accuracy test for the determination of Ligusticum striatum lactone I (n=6).

[0133]

[0134] The experimental results showed that the average recovery rate was 99.46% and the RSD was <1.2%, indicating that the method has good accuracy in determining the content of ligustrolide I.

[0135] Table 19 Results of the accuracy test for the determination of salvianolic acid B content (n=6).

[0136]

[0137] The experimental results showed that the average recovery rate was 100.46% and the RSD was <1.0%, indicating that the method has good accuracy in determining the content of salvianolic acid B.

[0138] Example 3: Establishment of the One-Measure-Multiple-Assessment Method

[0139] ① Determination of correction factor

[0140] In the standard curve Y = aX + b, X = (Y - b) / a = Y / a - b / a. Since the value of b is usually caused by error, when the value of a / b is greater than 100, the value of b / a can be ignored. In this case, X = Y / a can be directly calculated. Based on the standard curves of each component of Shechuan Naolitong granules, the slope correction method is used to calculate the relative correction factor. The formula for calculating the relative correction factor is fs / i = fs / fi; the formula for calculating the content is: Ci = Cs * Ai * fi / s / As. In the formula, fi is the slope of the analyte reference standard, fs is the slope of the reference standard, Ai is the peak area of ​​the analyte, As is the peak area of ​​the reference standard, Ci is the concentration of the analyte, and Cs is the concentration of the reference standard. The relative correction factors of genipin glycoside, chlorogenic acid, vasiloxane flavonoid glycoside, ferulic acid, and ligusticum lactone I relative to salvianolic acid B were 1.0584, 0.5943, 0.6433, 0.3427, and 0.2628, respectively.

[0141] ② Determination of relative retention time

[0142] The chromatographic peak of salvianolic acid B was located using a reference standard, while other analytes were located using their relative retention times to salvianolic acid B. When using a single-analyte-multiple-evaluation method, the relative retention times of other analyte peaks were calculated based on the retention time of salvianolic acid B. The results showed that the relative retention times of genipinic acid, chlorogenic acid, vaccaria segetalis flavonoids, ferulic acid, and ligusticum lactone I relative to salvianolic acid B were 0.250, 0.383, 0.496, 0.642, and 0.823, respectively.

[0143] ③ Evaluation of the robustness of relative correction factor and relative retention time

[0144] The effects were investigated using two different high-performance liquid chromatographs (Aglient 1260, Shimadzu LC-2010 HT) and three different Kromasil 100-5-C columns. 18 (250 mm×4.6 mm, 5 μm), Agilent Zorbax SB-Aq C 18 ((250 mm × 4.6 mm, 5 μm); Welch Ultimate LP-C 18 The effects of (250 mm × 4.6 mm, 5 μm) on the relative correction factor and relative retention time of each component are shown in Tables 20-21.

[0145] Table 20 Results of the durability test of the correction factor.

[0146]

[0147] The results show that the relative correction factor RSD of each component is less than 3.0%, which proves that the relative correction factor of each component has good robustness.

[0148] Table 21 Results of the relative retention time durability test.

[0149]

[0150] The results showed that the relative retention time RSD of each component was <3.0%, and the relative retention time of each component was within ±5% of the specified value, proving that the relative retention time of each component was durable.

[0151] ④ Comparison of results between the one-test-multiple-evaluation method and the external standard method

[0152] The contents of genipinic acid, chlorogenic acid, vasopressin, ferulic acid, ligustrazine I, and salvianolic acid B in 15 batches of muskheri granules were calculated using the external standard method and the one-test-multiple-evaluation method, respectively. The comparison results of the external standard method and the one-test-multiple-evaluation method for 15 batches of muskheri granules are shown in Table 21.

[0153] Table 21 Comparison of the results of calculating the contents of genipinic acid, chlorogenic acid, Wangbuliuxing flavonoid glycoside, ferulic acid, chuanxiong lactone I, and tanshinone B in 15 batches of Shechuan Naolitong granules using the external standard method and the one-test-multiple-evaluation method.

[0154]

[0155] Experimental conclusion:

[0156] "Shechuan Naolitong Granules" uses a multi-test method to simultaneously determine the content of genipin glycosides, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, chuanxiong lactone I, and danshen acid B. Compared with the original quality standard which uses thin-layer chromatography for qualitative identification, the detected index components are more quantitative and comprehensive, allowing for more comprehensive control of the finished product quality.

Claims

1. A fingerprint spectrum detection method for Shechuan Naolitong granules, characterized in that, The detection method includes the following steps: (1) Preparation of test solution: Take the powder of muskhozane granules, add 65-75% methanol, seal tightly, weigh, sonicate, cool, make up the loss, shake well, filter, evaporate to dryness, dissolve the residue in 65-75% methanol, make up to volume, shake well, filter, and take the filtrate to obtain the test solution. (2) Preparation of mixed reference solution: Accurately weigh appropriate amounts of geniposide, chlorogenic acid, limonene flavonoids, ferulic acid, ligustrazine lactone, and salvianolic acid B reference standards, and dissolve them in 70% methanol to prepare a mixed reference solution. The concentration of geniposide is 150-200 μg / mL, the concentration of chlorogenic acid is 150-170 μg / mL, the concentration of limonene flavonoids is 70-90 μg / mL, the concentration of ferulic acid is 130-170 μg / mL, the concentration of ligustrazine lactone is 160-190 μg / mL, and the concentration of salvianolic acid B is 700-740 μg / mL. Mix thoroughly to obtain the solution. (3) Chromatographic conditions: Column C 18 The mobile phase consisted of acetonitrile (phase A) and 0.1% phosphoric acid aqueous solution (phase B). The gradient elution ratios were as follows: 0–7 min, mobile phase A 5%, mobile phase B 95%; 7–12 min, mobile phase A 5%–10%, mobile phase B 95%–90%; 12–40 min, mobile phase A 10%–22%, mobile phase B 90%–78%; 40–60 min, mobile phase A 22%–25%, mobile phase B 78%–75%; 60–80 min, mobile phase A 25%–45%, mobile phase B 75%–55%; 80–85 min, mobile phase A 45%–70%, mobile phase B 55%–30%. 85–95 min, mobile phase A 70%–90%, mobile phase B 30%–10%; 95–105 min, mobile phase A 90%, mobile phase B 10%; 105–110 min, mobile phase A 90%–5%, mobile phase B 10%–95%; 110–120 min, mobile phase A 5%, mobile phase B 95%; detection wavelength 260–300 nm, column temperature 25–32 °C; (4) Establish fingerprint spectrum: Take the test solution from step (1) and the mixed reference solution from step (2), and determine them using the chromatographic conditions in step (3). Perform the detection according to the chromatographic conditions in step (3), and analyze multiple batches of the test solution of Shechuan Naolitong Granules. Import the samples into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to generate a reference fingerprint spectrum.

2. The detection method as described in claim 1, characterized in that, The detection method step (1) uses an ultrasonic frequency of 35-45kHz, an ultrasonic power of 350-450W, and an ultrasonic processing time of 20-40min.

3. The detection method as described in claim 1, characterized in that, In step (2) of the detection method, the concentration of methanol is 70%.

4. The detection method as described in claim 1, characterized in that, In step (2) of the detection method, the concentration of the mixed reference solution is 184.0 μg / mL for genipinic acid, 172.0 μg / mL for chlorogenic acid, 78.2 μg / mL for vaccaria segetalis flavonoid glycoside, 153.2 μg / mL for ferulic acid, 178.4 μg / mL for ligusticum lactone, and 722.0 μg / mL for salvianolic acid B.

5. The detection method as described in claim 1, characterized in that, The detection method step (3) involves a chromatographic column C. 18 The model number is Kromasil 100-5, and the column specifications are 250 mm × 4.6 mm and 5 μm.

6. The detection method as described in claim 1, characterized in that, The volumetric flow rate in step (3) of the detection method is 1.0 mL / min, the detection wavelength is 254 nm and 280 nm, and the column temperature is 25 °C.

7. The fingerprint pattern detection method as described in claim 1, characterized in that, The test solution is tested according to the chromatographic conditions in step (3), and the generated liquid chromatographic peak is compared with the reference fingerprint spectrum. The similarity is between 0.90 and 1.

00.

8. The detection method as described in claim 1, characterized in that, The detection method is a multi-evaluation content detection method, which includes the following steps: the preparation of the reference standard and the test solution and the chromatographic conditions are the same as those in steps (1) to (3) of claim 1. Danshensu B is used as the reference peak to determine the contents of genipin glycoside, chlorogenic acid, Wangbuliuxing flavonoid glycoside, ferulic acid and chuanxiong lactone I.

9. The detection method as described in claim 8, characterized in that, The chromatographic conditions for determining the contents of genipin, chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, ligustrazine lactone I, and salvianolic acid B were as follows: flow rate 1.0 mL / min, column temperature 30℃, injection volume 10 μL, wherein the detection wavelength of genipin was 254 nm, and the detection wavelengths of chlorogenic acid, Wangbuliuxing flavonoid glycosides, ferulic acid, ligustrazine lactone I, and salvianolic acid B were 280 nm.

10. The detection method according to claims 1 to 9, characterized in that, The detection method described herein is used for the quality detection of Shechuan Naolitong Granules, and for the identification of intermediates and effective components of traditional Chinese medicine.

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