Construction method of UPLC characteristic spectrum of comfrey and application thereof

Abstract

The application discloses a construction method of a UPLC characteristic spectrum of a radix-lithospermi and application thereof, 1) preparation of a control medicinal material reference solution; 2) preparation of a control reference solution: a certain amount of protocatechuic acid, protocatechuic aldehyde, ferulic acid, rosmarinic acid, caffeic acid tetramer and danshensu are taken to prepare a mixed solution by adding methanol, serving as the control reference solution; 3) preparation of a test solution; 4) determination: the control medicinal material reference solution, the control reference solution and the test solution are respectively taken and injected into a liquid chromatograph to determine characteristic spectra, wherein the characteristic spectra include at least 12 characteristic peaks; 5) qualitative analysis of a sample to be determined is carried out by using the characteristic spectra obtained in the above steps. The characteristic spectrum includes 12 characteristic peaks, can effectively distinguish radix-lithospermi (Xinjiang radix-lithospermi) and its pseudo products of different origins, and embodies the integrity of quality control of radix-lithospermi.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality analysis and testing technology, and in particular to a method for constructing UPLC characteristic maps of Lithospermum erythrorhizon and its application. Background Technology

[0002] Commercially available purple gromwell (Lithospermum erythrorhizon) is derived from the dried root of *Arnebia euchroma* (Royle) Johnst., a plant in the Boraginaceae family. It is harvested in spring and autumn, cleaned of mud and sand, and then dried. Purple gromwell was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It is bitter and cold in nature. It is used to treat evil qi in the heart and abdomen, jaundice, tonify the middle jiao and replenish qi, benefit the nine orifices, and clear the urinary tract. The *Flora of China* records that *Arnebia euchroma* (Royle) Johnst. (soft purple gromwell) is mainly produced in Xinjiang and Tibet, typically growing on sunny, gravelly slopes, alluvial fans, grasslands, and meadows at altitudes of 2100–3000 m. It is considered the highest quality purple gromwell medicinal material and has become the main source of commercial purple gromwell.

[0003] The main chemical components of Lithospermum erythrorhizon in Xinjiang include naphthoquinones, phytohexenes, phytohexenes, phenolic acids, alkaloids, and polysaccharides (Qian Xue, Li Haitao, Zeng Wanxiang, et al. Research progress on chemical components, pharmacological effects and product applications of Lithospermum erythrorhizon [J]. Chinese Wild Plant Resources, 2021, 40(3):52-56). Modern pharmacological studies have shown that it has the effects of clearing heat and blood, promoting blood circulation and detoxifying, and promoting rash eruption and eliminating spots. It can be used to treat various pharmacological activities such as blood heat and toxicity, purplish-black spots, measles that fail to erupt, sores, eczema, and burns.

[0004] Huang Rui et al. used UPLC chromatography to determine the content of naphthoquinones in wild and cultivated Lithospermum erythrorhizon from Xinjiang; at the same time, they used PCA and OPLS-DA, two chemical pattern recognition techniques, to determine that isobutyrylshikonin and β,β'-dimethylacryloylacrylamine could distinguish between wild and cultivated Lithospermum erythrorhizon from Xinjiang (Huang Rui, Dai Shengyun, Wu Dongxue, et al. Comparative study on the quality of wild and cultivated Lithospermum erythrorhizon from Xinjiang [J]. Journal of Pharmaceutical Analysis, 2024, 44(5):783-795.). Hu Hezhula et al. analyzed and predicted the Q-Marker of Lithospermum erythrorhizon, and found that naphthoquinones and polysaccharides could be candidate components for the classification Q-Marker of Lithospermum erythrorhizon (Hu Hezhula, Alatan Chaolumen, Liao Cuiping, et al. Research progress of Lithospermum erythrorhizon and prediction of quality markers [J]. Journal of Traditional Chinese Medicine, 2024, 52(6):105-110.). The roots of various medicinal plants in the Boraginaceae family contain naphthoquinone-like purple components and are often used interchangeably with other medicinal plants, such as Lithospermum erythrorhizon. This has led to the influx of imported Lithospermum erythrorhizon with unclear origins into the market, resulting in inconsistent quality and severely impacting product quality and efficacy. Existing literature largely focuses on the lipid-soluble naphthoquinone components, with limited research on water-soluble components. A comprehensive quality control method is lacking, and only a few studies have included fingerprint analysis of Lithospermum erythrorhizon medicinal materials.

[0005] Ding Wenhuan et al. conducted morphological and HPLC fingerprint analysis on Xinjiang Lithospermum and Lithospermum chrysanthemum, and found 5 common peaks with similarity greater than 0.9. (Ding Wenhuan, Li Jie, Zhang Xuejia, et al. Establishment of HPLC fingerprint and chemical pattern recognition analysis of Xinjiang Lithospermum and Lithospermum chrysanthemum [J]. Chinese Traditional and Herbal Drugs, 2022, 44(10):3220-3224.). Huang Rui et al. established HPLC content determination and fingerprint analysis method for Lithospermum herb, identified 9 common peaks, and determined the content of 6 naphthoquinone chemical components. The results showed that the similarity of 50 batches of Lithospermum herb varied greatly. (Huang Rui, Dai Shengyun, Wu Dongxue, et al. Quality evaluation of Lithospermum based on HPLC fingerprint and multi-index component content determination [J]. Chinese Pharmaceutical Journal, 2024, 59(10):929-937.). Through patent searches, Minzu University of China disclosed a method and application for establishing a fingerprint spectrum of Xinjiang Lithospermum based on HPCE. The capillary electrophoresis spectrum includes six characteristic peaks, used to distinguish Xinjiang Lithospermum from Inner Mongolia Lithospermum (Minzu University of China. Method and application for establishing a fingerprint spectrum of Xinjiang Lithospermum based on HPCE: CN201710007836.8[P]. 2020-08-04.). The fingerprint spectrum established in the above literature has a relatively small number of characteristic peaks and cannot simultaneously identify different origins of Lithospermum and genuine and counterfeit products, thus failing to comprehensively reflect the quality level of Xinjiang Lithospermum.

[0006] Therefore, it is necessary to develop new technologies to effectively distinguish between different types of comfrey and its counterfeits, and to control the overall quality of comfrey products. Summary of the Invention

[0007] Purpose of the invention: To address the shortcomings and defects of existing technologies, this invention provides a method for constructing a UPLC characteristic spectrum of Lithospermum erythrorhizon and its application. The characteristic spectrum contains 12 characteristic peaks, which can effectively distinguish Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) from different origins and its counterfeits. This reflects the holistic nature of Lithospermum erythrorhizon quality control, especially the overall evaluation and control of the quality of intermediates and finished products in each stage of the production process of the formulated granules.

[0008] Technical solution: The present invention provides a method for constructing a UPLC feature map of Lithospermum erythrorhizon, characterized by the following steps:

[0009] 1) Preparation of reference solution for reference medicinal material: Take Xinjiang Lithospermum erythrorhizon reference medicinal material, add water, heat and extract, filter, treat the filtrate with hydrochloric acid solution, add ethyl acetate and shake to extract, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol to obtain the solution;

[0010] 2) Preparation of reference solution: Take the set amount of protocatechuic acid reference standard, protocatechuic aldehyde reference standard, ferulic acid reference standard, rosmarinic acid reference standard, caffeic acid tetramer reference standard, and tanshinone reference standard, add methanol to prepare a mixed solution, which is used as the reference solution.

[0011] 3) Preparation of test solution: Take the Lithospermum erythrorhizon sample, extract with water, filter, treat the filtrate with hydrochloric acid solution, extract with ethyl acetate by shaking, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol to obtain the test solution;

[0012] 4) Determination: The reference solution of Xinjiang Lithospermum erythrorhizon, the reference standard solution, and the test solution were injected into the liquid chromatograph for determination, and the characteristic chromatograms were obtained, which included at least 12 characteristic peaks.

[0013] 5) Use the characteristic spectra obtained in the above steps to perform qualitative analysis of the sample to be tested.

[0014] In step 1), the volume of water added to the preparation of the reference solution of Xinjiang Lithospermum erythrorhizon is 25ml to 100ml, the extraction time is 30min to 120min, the volume concentration of hydrochloric acid solution is 0.5% to 10%, the pH of the solution after treatment is 1 to 2, and the extraction with ethyl acetate is performed 1 to 3 times, with an extraction volume of 20ml to 100ml each time.

[0015] In step 2), methanol is added to prepare a methanol aqueous solution with a volume concentration of 5% to 70%.

[0016] In step 3), the volume of water added is 15ml to 50ml, the extraction time is 15min to 60min, the volume concentration of hydrochloric acid solution added is 0.5% to 10%, the pH of the treated solution is 1 to 2, and the number of extractions with ethyl acetate is 1 to 3, with each extraction volume being 20ml to 100ml.

[0017] In step 4), the mobile phase A of the liquid chromatograph is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with a volume concentration of 0.05% to 0.2%. The chromatographic column used in the determination is packed with octadecylsilane-bonded silica gel. Gradient elution is adopted: 0 to 20 min, 4% A → 15% A, 20 min to 30 min, 15% A → 20% A, 30 min to 33 min, 20% A.

[0018] In step 4), the detection wavelength is 254nm to 300nm; the flow rate is 0.30ml to 0.40ml per minute; the column temperature is 35℃ to 43℃; and an ultraviolet detector is used.

[0019] In step 4), the ultra-high performance liquid chromatography (UHPLC) chromatogram of the Xinjiang Lithospermum sample has 12 characteristic peaks, which should correspond to the retention times of the 12 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 2, 4, 6, 11, and 12 should correspond to the retention times of the corresponding reference standard peaks. The peak corresponding to the 4-hydroxybenzoic acid reference standard peak is peak S1; calculate the relative retention times of peaks 3 and 5 with peak S1. The peak corresponding to the caffeic acid tetramer (Rabdosiin) reference standard peak is peak S2; calculate the relative retention times of peaks 7, 8, 9, and 10 with peak S2. All relative retention times should be within ±10% of the specified values, which are: 0.92 (peak 3), 2.25 (peak 5), 0.74 (peak 7), 0.88 (peak 8), 0.90 (peak 9), and 0.98 (peak 10).

[0020] The application of the method for constructing the UPLC characteristic map of Lithospermum erythrorhizon of the present invention in distinguishing different original Lithospermum erythrorhizon samples.

[0021] The application of the construction method of the present invention in distinguishing between Lithospermum erythrorhizon and its counterfeit samples.

[0022] The method for constructing the UPLC characteristic spectrum of Lithospermum erythrorhizon of the present invention can be applied to Lithospermum erythrorhizon medicinal materials, Lithospermum erythrorhizon slices, Lithospermum erythrorhizon standard decoctions, Lithospermum erythrorhizon preparation intermediates or finished Lithospermum erythrorhizon preparations.

[0023] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention provides a simple and accurate characteristic chromatographic method for distinguishing different types of Lithospermum erythrorhizon (Xinjiang Lithospermum) and its adulterants. The characteristic chromatogram contains 12 characteristic peaks, which can effectively distinguish different types of Lithospermum erythrorhizon (Xinjiang Lithospermum) and its adulterants, reflecting the holistic nature of Lithospermum erythrorhizon quality control, especially the overall evaluation and control of intermediate and finished product quality at each stage of the production process of the formulated granules. This ensures the quality stability of the finished product, better guides production, and guarantees the quality and efficacy of the product.

[0024] The method of this invention features short detection time, simple operation, low solvent consumption, and minimal environmental pollution. After methodological validation, it demonstrates good reproducibility and stability, reliable recovery rate, low detection cost, and high detection efficiency. This invention provides a new analytical tool for the quality control of Lithospermum erythrorhizon (Xinjiang Lithospermum), and can distinguish different original sources and their adulterants, providing important assurance for the standardized production of Lithospermum erythrorhizon (Xinjiang Lithospermum) preparations. The characteristic chromatogram of Lithospermum erythrorhizon established by this invention has 12 characteristic peaks, presenting rich chromatographic information containing multiple chemical components, enabling comprehensive quality control of Lithospermum erythrorhizon (Xinjiang Lithospermum) formulation granules. Attached Figure Description

[0025] Figure 1The image shows the characteristic chromatogram of the formula granules of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon); Peak 1: Tanshinone; Peak 2: Protocatechuic acid; Peak 3: Protocatechuic aldehyde; Peak 4 (S1): 4-hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Rabdosiin; Peak 12: Rosmarinic acid;

[0026] Figure 2 The image shows the characteristic spectrum of intermediates of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon); Peak 1: Tanshinone; Peak 2: Protocatechuic acid; Peak 3: Protocatechuic aldehyde; Peak 4 (S1): 4-hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Rabdosiin; Peak 12: Rosmarinic acid;

[0027] Figure 3 The chromatogram shows the characteristic properties of the standard decoction of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon); Peak 1: Tanshinone; Peak 2: Protocatechuic acid; Peak 3: Protocatechuic aldehyde; Peak 4 (S1): 4-hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Rabdosiin; Peak 12: Rosmarinic acid.

[0028] Figure 4 Characteristic chromatogram of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) slices; Peak 1: Tanshinone; Peak 2: Protocatechuic acid; Peak 3: Protocatechuic aldehyde; Peak 4 (S1): 4-hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Rabdosiin; Peak 12: Rosmarinic acid;

[0029] Figure 5 This is a characteristic spectrum of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon); Peak 1: Tanshinone; Peak 2: Protocatechuic acid; Peak 3: Protocatechuic aldehyde; Peak 4 (S1): 4-hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Rabdosiin; Peak 12: Rosmarinic acid;

[0030] Figure 6 Chromatograms of reference standard, reference medicinal material, and Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules;

[0031] Figure 7 UPLC chromatograms of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formulation granules at different detection wavelengths;

[0032] Figure 8 UPLC plots for different extraction solvents;

[0033] Figure 9 UPLC chromatograms for pH testing;

[0034] Figure 10 UPLC diagrams for different extraction methods;

[0035] Figure 11 UPLC plots for different extraction volumes;

[0036] Figure 12 UPLC plots for different extraction times;

[0037] Figure 13 UPLC plots for different extraction times;

[0038] Figure 14 UPLC plots for different extraction solvents;

[0039] Figure 15 Chromatographic column analysis of the characteristic chromatogram of Lithospermum erythrorhizon formula granules;

[0040] Figure 16 Column temperature analysis of the characteristic spectrum of Lithospermum erythrorhizon formula granules;

[0041] Figure 17 Flow rate analysis of the characteristic spectrum of Lithospermum erythrorhizon formula granules;

[0042] Figure 18 Comparative chromatograms of characteristic features of standard decoctions from different sources of Lithospermum erythrorhizon;

[0043] Figure 19 Comparative chart of characteristic features of genuine and counterfeit Lithospermum erythrorhizon decoctions;

[0044] Figure 20 A diagram showing the specificity test results for the formula granules of Lithospermum erythrorhizon;

[0045] Figure 21 This is a diagram illustrating the overall integrity of the purple gromwell granule formula. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0047] The method for constructing the UPLC feature map of Lithospermum erythrorhizon of the present invention includes the following steps:

[0048] 1) Select samples of Lithospermum erythrorhizon and prepare test solutions; use Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) as the reference herb and prepare reference solutions of the reference herb; use tanshinone, protocatechuic acid, protocatechuic aldehyde, 4-hydroxybenzoic acid, ferulic acid, caffeic acid tetramer (Rabdosiin), and rosmarinic acid as reference standards and prepare reference solutions of the reference standards respectively.

[0049] Preparation of reference solution: Take 0.5g-2g of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) reference material, place it in a stoppered conical flask, add 25ml-100ml of water, heat under reflux for 30-120 minutes, cool, shake well, filter, adjust the pH of the filtrate to 1-2 with 0.5%-10% hydrochloric acid solution, extract with ethyl acetate 1-3 times, 20ml-100ml each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in methanol, and use as the reference solution of the reference material. Preparation of reference solutions: Take appropriate amounts of protocatechuic acid reference standard, 4-hydroxybenzoic acid reference standard, ferulic acid reference standard, and rosmarinic acid reference standard, accurately weigh them, and add methanol to prepare solutions containing 15-40 μg of protocatechuic acid reference standard, 4-hydroxybenzoic acid reference standard, ferulic acid reference standard, and rosmarinic acid per 1 ml, and 20-80 μg of sodium tanshinone (equivalent to 18-72 μg of tanshinone per 1 ml) as reference solutions.

[0050] Preparation of the test solution: Take 0.2g to 1.0g of the formula granules of Lithospermum erythrorhizon, grind them into a fine powder, place them in a stoppered conical flask, add 15ml to 50ml of water, seal tightly, and sonicate (power 250W, frequency 40kHz) for 15 to 60 minutes. Cool, shake well, filter, and adjust the pH of the filtrate to 1 to 2 with 0.5% to 10% hydrochloric acid solution. Then extract with ethyl acetate by shaking 1 to 3 times, 20mL to 100mL each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in methanol to obtain the test solution.

[0051] Preferably, the extractant for the reference solution is an aqueous methanol solution with a volume concentration of 5% to 70%.

[0052] 2) Perform ultra-high performance liquid chromatography (UHPLC) analysis on both the reference solution and the test solution to construct the characteristic chromatogram of the sample, such as... Figure 1 As shown, the characteristic spectrum of the formula granules of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) includes at least 12 characteristic peaks. Among the characteristic peaks, there are reference peaks that correspond to the reference standards of tanshinone, protocatechuic acid, protocatechuic aldehyde, 4-hydroxybenzoic acid, ferulic acid, caffeic acid tetramer (Rabdosiin), and rosmarinic acid.

[0053] Perform ultra-high performance liquid chromatography (UHPLC) analysis on the test solution and reference solution prepared in step 1) to obtain the corresponding UHPLC chromatograms; accurately pipette 0.5 μl to 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the results.

[0054] Chromatographic conditions: The column was packed with octadecylsilane-bonded silica gel; acetonitrile was used as mobile phase A, and 0.05%–0.2% phosphoric acid solution was used as mobile phase B, with gradient elution; the flow rate was 0.30–0.40 ml per minute; the column temperature was 35℃–43℃; an ultraviolet detector was used; and the detection wavelength was 254–300 nm.

[0055] Preferably, octadecylsilane-bonded silica gel is used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the specifications in the table below; the flow rate is 0.35 ml per minute; the column temperature is 40 °C; and the detection wavelength is 270 nm.

[0056]

[0057] Optionally, the Lithospermum erythrorhizon (Xinjiang Lithospermum) formula granules can be replaced with other Lithospermum erythrorhizon (Xinjiang Lithospermum) samples, such as Lithospermum erythrorhizon (Xinjiang Lithospermum) medicinal materials, Lithospermum erythrorhizon (Xinjiang Lithospermum) slices, Lithospermum erythrorhizon (Xinjiang Lithospermum) standard decoction, Lithospermum erythrorhizon (Xinjiang Lithospermum) preparation intermediates or Lithospermum erythrorhizon (Xinjiang Lithospermum) preparation finished products.

[0058] Selecting the intermediate of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) as the ... sample, repeating step 1), the constructed Lithospermum eryth Figure 2 As shown.

[0059] Selecting the standard decoction of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) as the sample, and repeating step 1), the characteristic spectrum of the constructed standard decoction of Lithospermum erythrorhizon is as follows. Figure 3 As shown

[0060] Selecting Lithospermum erythrorhizon (Xinjiang Lithospermum) as the Lithospermum erythrorhizon sample, repeating step 1), the constructed Lithospermum erythrorhizon (Xinjiang Lithospermum) characteristic spectrum is as follows. Figure 4 As shown.

[0061] Select Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) slices as the Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) sample, repeat step 1), and construct the characteristic spectrum of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) slices as follows. Figure 5 As shown.

[0062] like Figure 6 The chromatogram of the formula granules of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) should show 12 characteristic peaks, which should correspond to the retention times of the 12 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1, 2, 4, 6, 11, and 12 should correspond to the retention times of the corresponding reference standard peaks. The peak corresponding to the 4-hydroxybenzoic acid reference standard is peak S1. Calculate the relative retention times of peaks 3 and 5 with peak S1. The peak corresponding to the caffeic acid tetramer (Rabdosiin) reference standard is peak S2. Calculate the relative retention times of peaks 7, 8, 9, and 10 with peak S2. The relative retention times should be within ±10% of the specified values, which are: 0.92 (peak 3), 2.25 (peak 5), 0.74 (peak 7), 0.88 (peak 8), 0.90 (peak 9), and 0.98 (peak 10).

[0063] 3) Using the characteristic spectra obtained in step 2), the original source of the sample to be tested is identified; a comparison of the characteristic spectra of Xinjiang Lithospermum and Inner Mongolia Lithospermum is shown below. Figure 18 As shown, characteristic peaks 6, 7, and 11 in Xinjiang Lithospermum were not detected in Inner Mongolia Lithospermum, while unique peak A was present in Inner Mongolia Lithospermum.

[0064] 4) Using the characteristic spectra obtained in step 2), the authenticity of the sample to be tested is determined. A comparison of the characteristic spectra of Lithospermum erythrorhizon (Xinjiang Lithospermum) and its counterfeits (Lithospermum erythrorhizon, Potentilla chinensis) is shown below. Figure 19 As shown, there are significant differences between Lithospermum and its counterfeit products. In Lithospermum erythrorhizon, only peaks 4 and 10 from Lithospermum erythrorhizon were detected, with peak a being the unique peak. In Potentilla chinensis, peaks 2, 4, 10, and 12 from Lithospermum erythrorhizon were detected, with peaks A, B, C, D, and E being the unique peaks.

[0065] The characteristic spectrum detection method for Lithospermum erythrorhizon samples provided by this invention can simultaneously identify different origins and genuine / counterfeit products. This method is convenient, rapid, highly specific, and stable. By implementing quality control from multiple aspects, including characteristic spectrum analysis, origin identification, and genuine / counterfeit product identification, it provides stronger assurance for the industrialization of Lithospermum erythrorhizon formula granules.

[0066] Example 1:

[0067] Detection and establishment of reference chromatograms for Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules, standard decoctions, and medicinal materials / processed slices:

[0068] Three batches of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules, nine batches of Lithospermum erythrorhizon standard decoction, and nine batches of Lithospermum erythrorhizon medicinal materials / slices were taken as Lithospermum erythrorhizon samples, and quality control tests were carried out according to the following steps:

[0069] 1) Select the formula granules of Lithospermum erythrorhizon and prepare the test solution; use Lithospermum erythrorhizon as the reference herb and prepare the reference solution of the reference herb; use tanshinone, protocatechuic acid, protocatechuic aldehyde, 4-hydroxybenzoic acid, ferulic acid, caffeic acid tetramer (Rabdosiin), and rosmarinic acid as reference standards and prepare reference solutions of reference standards respectively.

[0070] Preparation of reference solution: Take 1g of Lithospermum erythrorhizon reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60 minutes, cool, shake well, filter, add 2% hydrochloric acid solution to adjust the pH value to 1-2, extract twice with ethyl acetate, 50mL each time, combine the ethyl acetate extracts, evaporate to dryness, add 3ml of methanol to dissolve the residue, and use it as the reference solution for the reference material.

[0071] Preparation of reference solution: Take appropriate amounts of tanshinone, protocatechuic acid, protocatechuic aldehyde, 4-hydroxybenzoic acid, ferulic acid, caffeic acid tetramer (Rabdosiin), and rosmarinic acid reference standards, accurately weigh them, and add 10% methanol to prepare a mixed solution containing 20 μg of each of the following reference standards per ml: protocatechuic acid, 4-hydroxybenzoic acid, ferulic acid, and rosmarinic acid, and 40 μg of tanshinone sodium reference standard per ml (equivalent to 36 μg of tanshinone per ml). This solution is used as the reference solution.

[0072] Preparation of test solution of Lithospermum erythrorhizon formula granules: Take about 0.5g of Lithospermum erythrorhizon formula granules, place them in a stoppered conical flask, add 25ml of water, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, add 2% hydrochloric acid solution to adjust the pH value to 1-2, extract twice with ethyl acetate, 25mL each time, combine the ethyl acetate solutions, evaporate to dryness, add 3ml of methanol to dissolve the residue, and the test solution is obtained.

[0073] Preparation of the test solution of the standard decoction of Lithospermum erythrorhizon: Take about 0.2g of standard decoction powder, place it in a stoppered conical flask, add 25ml of water, stopper tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, add 2% hydrochloric acid solution to adjust the pH value to 1-2, extract twice with ethyl acetate, 25mL each time, combine the ethyl acetate solutions, evaporate to dryness, add 3ml of methanol to dissolve the residue, and the test solution is obtained.

[0074] Preparation of test solution of purple herb / processed medicinal material: Take 1g of purple herb / processed medicinal material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60 minutes, cool, shake well, filter, adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution, extract twice with ethyl acetate, 50ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 3ml of methanol, and the test solution is obtained.

[0075] 2) Perform ultra-high performance liquid chromatography (UHPLC) analysis on the test solution and reference solution prepared in step 1) to obtain the corresponding UHPLC chromatograms; accurately pipette 1 μl of the reference solution and the test solution into the liquid chromatograph and measure them to obtain the results.

[0076] Among them, the chromatographic conditions parameters are:

[0077] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 2.1 mm inner diameter, 1.6 μm particle size); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 0.35 mL / min; the column temperature was 40 °C; and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the rabdosiin tetramer peak, should be no less than 5000.

[0078]

[0079] like Figure 6 As shown, the characteristic chromatogram of the Lithospermum erythrorhizon formula granules should show 12 characteristic peaks in the chromatogram of the test sample, and these peaks should correspond to the retention times of the 12 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 2, 4, 6, 11, and 12 should correspond to the retention times of the corresponding reference standard peaks. The peak corresponding to the 4-hydroxybenzoic acid reference standard peak is peak S1. Calculate the relative retention times of peaks 3 and 5 with peak S1. The peak corresponding to the caffeic acid tetramer (Rabdosiin) reference standard peak is peak S2. Calculate the relative retention times of peaks 7, 8, 9, and 10 with peak S2. All relative retention times should be within ±10% of the specified values, which are: 0.92 (peak 3), 2.25 (peak 5), 0.74 (peak 7), 0.88 (peak 8), 0.90 (peak 9), and 0.98 (peak 10). The determination results are shown in Tables 1, 2, and 3 below.

[0080] Table 1. Determination results (relative retention time) of granulated samples of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon).

[0081]

[0082] Table 2. Determination results (relative retention time) of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) medicinal materials / processed slices.

[0083]

[0084] Table 3. Determination results (relative retention time) of standard decoction samples of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon).

[0085]

[0086] 3) Compare the ultra-high performance liquid chromatogram obtained in step 2) with the characteristic chromatogram in step 1) to analyze the sample;

[0087] The test results above show that, based on the analysis, the characteristic spectra of the three batches of Lithospermum erythrorhizon formula granules and the nine batches of Lithospermum erythrorhizon standard decoctions and medicinal materials / processed slices all meet the requirements of the characteristic spectra in step 2).

[0088] This embodiment is used to confirm the optimal scheme for establishing the characteristic spectrum of Lithospermum erythrorhizon sample. Specifically, it includes comparing the schemes for chromatographic conditions, wavelength selection, and sample solution preparation, and selecting the optimal chromatographic conditions and sample preparation scheme. The optimal scheme is then examined for specificity, integrity, precision, intermediate precision, stability, repeatability, and robustness.

[0089] Example 2:

[0090] Establishment and methodological study of the characteristic spectral method for Lithospermum erythrorhizon formula granules:

[0091] This embodiment is used to confirm the optimal scheme for establishing characteristic chromatograms in the quality control method of Lithospermum erythrorhizon formula granules. Specifically, it includes comparing the schemes of chromatographic conditions, wavelength selection, and test sample solution preparation, selecting the optimal chromatographic conditions and test sample preparation scheme; and examining the specificity, integrity, precision, intermediate precision, stability, repeatability, and robustness of the optimal scheme.

[0092] 1. Instruments and reagents:

[0093] The following equipment was used: Agilent Technologies 1290 Inftnity UHPLC; Thermo Vanquish UHPLC; KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 shaker (Jintan Kexing Instrument Factory). Pure water system (Millipore); AS165W centrifuge (Asia Pacific (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); methanol (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Aladdin); ultrapure water; and all other reagents were analytical grade.

[0094] The reference standards for tanshinone sodium, protocatechuic acid, 4-hydroxybenzoic acid, ferulic acid, rosmarinic acid, and protocatechuic aldehyde were all purchased from the National Institutes for Food and Drug Control, with batch numbers 110855-201915, 110809-201906, 101149-202204, 110773-201915, 111871-201505, and 110810-201909, respectively.

[0095] Rabdosiin reference standard was purchased from Chengdu Pusi Biotechnology Co., Ltd., batch number PS013125.

[0096] The reference material of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) was purchased from the National Institutes for Food and Drug Control, batch number 121430-201504.

[0097] The formula granules, intermediates, medicinal materials / processed slices, and standard decoctions of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) are all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0098] 2. Determination of detection wavelength:

[0099] Take about 0.5g of this product, place it in a stoppered conical flask, add 25ml of water, seal tightly, and sonicate (250W power, 40kHz frequency) for 30 minutes. Cool, shake well, and filter. Adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution. Extract twice with ethyl acetate, 25mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 3ml of methanol. Collect chromatograms of the sample at 254nm, 270nm, and 300nm, as shown below. Figure 7 As shown.

[0100] The results showed that the chromatographic peak information was rich and the response was good at a wavelength of 270 nm, so 270 nm was selected as the detection wavelength.

[0101] 3. Determination of chromatographic conditions:

[0102] The column was packed with octadecylsilane-bonded silica gel (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in the table below; the flow rate was 0.35 ml per minute; the column temperature was 40 °C; and the detection wavelength was 270 nm.

[0103]

[0104] 4. Preparation of the test solution:

[0105] 4.1 Investigation of different extraction solvents:

[0106] Take an appropriate amount of granulated Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon), grind it into a fine powder, and take about 0.5g. Divide the powder into five parallel portions, accurately weigh each portion, and place them in stoppered conical flasks. Add water, 30% methanol, 50% methanol, 80% methanol, and 25ml of methanol to each flask, respectively. Seal the flasks tightly and sonicate (250W power, 40kHz frequency) for 30 minutes. Cool, shake well, and filter. Adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution. Extract twice with ethyl acetate, 25mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 3ml of methanol. Accurately pipette 1μl of each test solution and inject it into the liquid chromatograph. Determine the chromatographic conditions as described above, and calculate the peak area / sample weight. The results are shown in Table 4. Figure 8 .

[0107] Table 4 Comparison of extraction efficiencies of different extraction solvents (peak area / sample weight)

[0108]

[0109] The results showed that water was the most efficient solvent for extraction, thus water was determined to be the optimal solvent.

[0110] 4.2 pH test:

[0111] Take an appropriate amount of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) granules, grind them finely, and take about 0.5g. Divide the mixture into two parallel groups (two portions per group), place each in a stoppered conical flask, add 25ml of water, seal tightly, and sonicate (250W power, 40kHz frequency) for 30 minutes. Cool, shake well, and filter. Adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution. Alternatively, extract twice directly with ethyl acetate (25mL each time) without pH adjustment. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 3ml of methanol. Accurately pipette 1μl of each test solution and inject it into the liquid chromatograph. Determine the chromatographic conditions as described above, calculate the peak area / sample weight, and the results are shown in Table 5. Figure 9 .

[0112] Table 5. pH Assessment of Characteristic Spectra (Peak Area / Sample Weight)

[0113]

[0114] The results showed that the characteristic peak response was higher when the pH was adjusted, therefore pH adjustment is necessary to determine the preparation method of the test sample.

[0115] 4.3 Examination of different extraction methods:

[0116] Take an appropriate amount of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) granules, grind them finely, and take about 0.5g. Divide the mixture into two parallel groups, with two replicates per group. Place each group in a stoppered conical flask, add 25ml of water, seal tightly, and treat with sonication, shaking, and reflux for 30 minutes respectively. Cool, shake well, and filter. Adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution. Extract twice with ethyl acetate, 25mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 3ml of methanol. Accurately pipette 1μl of each test solution and inject it into the liquid chromatograph. Determine the chromatographic conditions as described above, and calculate the peak area / sample weight. The results are shown in Table 6. Figure 10 .

[0117] Table 6. Examination of Feature Spectrum Extraction Methods (Peak Area / Sample Size)

[0118]

[0119] The results showed that the extraction efficiency of different extraction methods was not significantly different. Considering the simplicity of the experimental operation, ultrasonic treatment was selected as the extraction method.

[0120] 4.4 Investigation of different extraction volumes:

[0121] Take an appropriate amount of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) granules, grind them finely, and take about 0.5g. Divide the mixture into four parallel groups (two replicates per group), place each group in a stoppered conical flask, add 15ml, 25ml, and 50ml of water respectively, seal tightly, and sonicate (250W power, 40kHz frequency) for 30 minutes. Cool, shake well, and filter. Adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution. Extract twice with ethyl acetate, 25mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 3ml of methanol. Accurately pipette 1μl of each test solution and inject it into the liquid chromatograph. Determine the chromatographic conditions as described above, calculate the peak area of ​​the characteristic peak / sample weight, and the results are shown in Table 7. Figure 11 .

[0122] Table 7. Investigation of different extraction volumes (peak area / sample weight * volume)

[0123]

[0124] The results showed that the extraction efficiency was not significantly different with different extraction volumes, but the characteristic peak response was larger with 25 ml of extraction solvent. Therefore, 25 ml of extraction solvent was selected.

[0125] 4.5. Examination of different extraction times:

[0126] Take an appropriate amount of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) granules, grind them finely, take about 0.5g, divide into 4 parallel portions, place in a stoppered conical flask, add 25ml of water, seal tightly, and sonicate (power 250W, frequency 40kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively. Cool, shake well, filter, adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution, extract twice with ethyl acetate, 25mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 3ml of methanol, and obtain the final product. Accurately pipette 1μl of each test solution and inject it into the liquid chromatograph, determine the chromatographic conditions as described above, calculate the peak area of ​​the characteristic peak / sample weight, and the results are shown in Table 8. Figure 12 .

[0127] Table 8 Comparison of extraction efficiency at different extraction times (peak area / sample volume)

[0128]

[0129] The results showed that the extraction efficiency was high when ultrasonic treatment lasted for 30 minutes. Considering the need for complete extraction and time saving, the extraction time was determined to be 30 minutes.

[0130] 4.6 Investigation of different extraction times:

[0131] Take an appropriate amount of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) granules, grind them finely, and take about 0.5g. Divide the mixture into four parallel groups (two replicates per group), place each group in a stoppered conical flask, add 25ml of water, seal tightly, and sonicate (250W power, 40kHz frequency) for 30 minutes. Cool, shake well, and filter. Adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution. Extract with ethyl acetate once, twice, and three times, 25mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 3ml of methanol. Accurately pipette 1μl of each test solution and inject it into the liquid chromatograph. Determine the chromatographic conditions as described above, calculate the peak area of ​​the characteristic peak / sample weight, and the results are shown in Table 9. Figure 13 .

[0132] Table 9 Comparison of different extraction times (peak area / sample weight)

[0133]

[0134] The results showed that the extraction efficiency tended to stabilize after two extractions. Considering both sufficient extraction and time saving, two extractions were determined to be the optimal choice.

[0135] 4.7 Investigation of different extraction solvents:

[0136] Take an appropriate amount of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) granules, grind them finely, take about 0.5g, divide into 4 parallel groups (2 replicates per group), place in a stoppered conical flask, add 25ml of water, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, adjust the pH of the filtrate to 1-2 with 2% hydrochloric acid solution, extract twice with ethyl acetate and n-butanol respectively, 25mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 3ml of methanol, and obtain the final product. Accurately pipette 1μl of each test solution and inject it into the liquid chromatograph, determine the chromatographic conditions as described above, calculate the peak area of ​​the characteristic peak / sample weight, and the results are shown in Table 10. Figure 14 .

[0137] Table 10 Comparison of different extraction solvents (peak area / sample weight)

[0138]

[0139] The results showed that the extraction efficiency was higher when ethyl acetate was used as the extraction solvent, thus ethyl acetate was determined to be the optimal extraction solvent.

[0140] 4.8 Determination of the preparation method for the test solution:

[0141] Based on the above research results, the method for preparing the test solution of the characteristic spectrum of Lithospermum erythrorhizon formula granules is determined as follows:

[0142] Take an appropriate amount of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) granules, grind them into a fine powder, take about 0.5g, place them in a stoppered conical flask, add 25ml of water, seal tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Let it cool, shake well, filter, add 2% hydrochloric acid solution to the filtrate to adjust the pH to 1-2, extract twice with ethyl acetate, 25mL each time, combine the ethyl acetate extracts, evaporate to dryness, add 3ml of methanol to dissolve the residue, and the product is obtained.

[0143] 5. Research on the methodology of feature mapping:

[0144] 5.1 Specificity Examination:

[0145] The test solution of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules and the negative control solution lacking Lithospermum erythrorhizon were injected into the liquid chromatograph. The results are shown below. Figure 20 .

[0146] Experimental results show that the negative solution has no chromatographic peaks at the retention times of the characteristic peaks in the chromatogram of the test sample solution, indicating that the solvent and excipients do not interfere with the determination of Lithospermum erythrorhizon formula granules, and this method is specific for the determination of Lithospermum erythrorhizon formula granules.

[0147] 5.2 Overall Assessment:

[0148] Under the established chromatographic conditions, the elution time was extended to investigate whether residual impurity peaks would affect subsequent samples under the given chromatographic conditions. The results are shown below. Figure 21 .

[0149] The experimental results show that extending the elution time resulted in no impurity peaks, indicating that the chromatographic conditions basically met the principle of maximizing information content and had no impact on the analysis of subsequent samples.

[0150] 5.3 Precision test:

[0151] Take a batch of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules, prepare the test solution according to the test solution preparation method, inject 1 μl of the sample 6 times consecutively, record the retention time of the characteristic peak, calculate the relative retention time according to the requirements of the main text, and the results are shown in Table 11.

[0152] Table 11 Precision Experiment Results (Relative Retention Time)

[0153]

[0154] The results show that the relative retention time RSD of each characteristic peak is less than 1%, indicating good precision.

[0155] 5.4 Intermediate Precision Examination:

[0156] Take a batch of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules and prepare them into test solutions according to the test solution preparation method. Two experimenters, A and B, each prepared 3 samples according to the test solution preparation method. 1 μl of each sample was injected into an Agilent or Waters instrument, and the retention time of the characteristic peak was recorded. The relative retention time was calculated according to the requirements in the text. The results are shown in Table 12.

[0157] Table 12 Intermediate Precision Study (Relative Retention Time)

[0158]

[0159] The results show that the relative retention times of each characteristic peak are within the specified range, indicating good intermediate precision.

[0160] 5.5 Stability Test:

[0161] Take a batch of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules and prepare test solutions according to the test solution preparation method. Inject 1 μl of the solution at 0, 2, 4, 8, 12, 16 and 24 hours respectively, record the retention time of the characteristic peak, and calculate the relative retention time according to the requirements of the main text. The results are shown in Table 13.

[0162] Table 13 Stability Study (Relative Retention Time)

[0163]

[0164] The results showed that the relative retention time RSD of each characteristic peak was less than 1%, indicating that the test solution had good stability within 24 hours.

[0165] 5.6 Repeatability Test:

[0166] Take a batch of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules, make 6 parallel groups, prepare test solutions according to the test solution preparation method, inject 1 μl of each sample, record the retention time of the characteristic peak, calculate the relative retention time according to the requirements, and the results are shown in Table 14.

[0167] Table 14 Repeatability Tests (Relative Retention Times)

[0168]

[0169]

[0170] The results show that the relative retention time RSD of each characteristic peak is less than 1%, and the method has good repeatability.

[0171] 5.7 Durability Test:

[0172] 5.7.1 Column Investigation:

[0173] A batch of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules were taken, and test solutions were prepared according to the preparation method of test solution. Three chromatographic columns were used: Syncronis C18 (2.1 mm × 100 mm, 1.7 μm); GLOD aq (2.1 mm × 150 mm, 1.9 μm); and CORTECST3 (2.1 × 150 mm, 1.6 μm). 1 μl of each column was injected, and the corresponding chromatograms were recorded. The results are shown in Table 15. Figure 15 .

[0174] Table 15 Column Analysis (Relative Retention Times)

[0175]

[0176] The results showed that the Syncronis C18 (2.1 mm × 100 mm, 1.7 μm) column exhibited peak encapsulation. The GLOD aq (2.1 mm × 150 mm, 1.9 μm) and CORTECS T3 (2.1 × 150 mm, 1.6 μm) columns showed similar relative retention times and relative peak areas for each characteristic peak, indicating good resolution and robustness. Therefore, the CORTECS T3 (2.1 × 150 mm, 1.6 μm) column is recommended for the characteristic chromatographic analysis of Lithospermum erythrorhizon granules.

[0177] 5.7.2 Column Temperature Investigation:

[0178] A batch of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules were taken, and a test solution was prepared according to the preparation method of the test solution. The test solution was tested at three temperatures: 35℃, 40℃, and 43℃. 1 μl of the solution was injected at each temperature, and the retention time of the characteristic peak was recorded. The results are shown in Table 16. Figure 16 .

[0179] Table 16 Column Temperature Investigation (Relative Retention Time)

[0180]

[0181] The results showed that the column temperature range of 35℃ to 43℃ was relatively stable for the determination of the samples and all met the retention time requirements specified by the method, indicating good robustness.

[0182] 5.7.3 Flow velocity assessment:

[0183] A batch of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) formula granules were taken, and a test solution was prepared according to the preparation method of the test solution. The sample separation effect at three flow rates of 0.3 ml, 0.35 ml, and 0.4 ml per minute was investigated. The results are shown in Table 17. Figure 17 .

[0184] Table 17 Flow velocity analysis (relative retention time)

[0185]

[0186] The results showed that the sample determination results were relatively stable when the flow rate was in the range of 0.3 ml / min to 0.4 ml / min, and all met the retention time requirements specified by the method, indicating good robustness.

[0187] Example 3:

[0188] Identification of different origins of Lithospermum erythrorhizon:

[0189] 1. Instruments and reagents:

[0190] The following equipment was used: Agilent Technologies 1290 Inftnity UHPLC; Thermo Vanquish UHPLC; KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 shaker (Jintan Kexing Instrument Factory). Pure water system (Millipore); AS165W centrifuge (Asia Pacific (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); methanol (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Aladdin); ultrapure water; and all other reagents were analytical grade.

[0191] The standard decoctions of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) and Lithospermum erythrorhizon (Inner Mongolia Lithospermum erythrorhizon) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0192] 2. Determination of chromatographic conditions:

[0193] The column was packed with octadecylsilane-bonded silica gel (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in the table below; the flow rate was 0.35 ml per minute; the column temperature was 40 °C; and the detection wavelength was 270 nm.

[0194]

[0195] 3. Preparation of the test solution:

[0196] Take about 0.2g of sample powder, place it in a stoppered conical flask, add 25ml of water, seal tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, add 2% hydrochloric acid solution to adjust the pH to 1-2, extract twice with ethyl acetate, 25mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 3ml of methanol, and the sample is ready.

[0197] 4. Sample testing and data analysis:

[0198] Seven batches of Xinjiang Lithospermum erythrorhizon standard decoction and three batches of Inner Mongolia Lithospermum erythrorhizon standard decoction were taken respectively, and sample test solutions were prepared according to the above-mentioned test solution preparation method. 1 μl of each solution was accurately pipetted and injected into the liquid chromatograph. Under the above-mentioned chromatographic conditions, the characteristic chromatograms of Xinjiang Lithospermum erythrorhizon and Inner Mongolia Lithospermum erythrorhizon standard decoctions were determined respectively. The "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission was used to generate a reference characteristic chromatogram. The reference characteristic chromatograms of the two were then imported into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)" for comparative analysis.

[0199] The results are as follows Figure 18 As shown, some characteristic peaks in the standard decoction of Lithospermum erythrorhizon from Xinjiang were not detected in Lithospermum erythrorhizon from Inner Mongolia. Only 9 common characteristic peaks of Lithospermum erythrorhizon from Xinjiang were present. Peaks 6, 7, and 11 were not detected in the standard decoction of Lithospermum erythrorhizon from Inner Mongolia. Peak A is a unique peak of Lithospermum erythrorhizon from Inner Mongolia.

[0200] Further analysis of the differences between the standard decoctions of Xinjiang Lithospermum and Inner Mongolia Lithospermum was conducted using relative peak area analysis. Peak 4 was used as the reference peak to compare the differences in the relative peak areas of each characteristic peak in the standard decoctions of the two countries. The results are shown in Tables 18 and 19.

[0201] Table 18 Relative peak areas of Xinjiang Lithospermum and standard decoctions of different origins (peak 4: S peak)

[0202]

[0203]

[0204] Table 19. Differences in relative peak area between standard decoctions of Lithospermum erythrorhizon (Xinjiang Lithospermum) and Lithospermum erythrorhizon (Inner Mongolia Lithospermum).

[0205]

[0206] The results show that the relative peak area ranges of peaks 9, 10, and 12 do not overlap between Xinjiang and Inner Mongolian Lithospermum erythrorhizon, and the established characteristic method can effectively distinguish different Lithospermum erythrorhizon samples.

[0207] Example 4:

[0208] Identification of genuine and counterfeit Lithospermum erythrorhizon:

[0209] 1. Instruments and reagents:

[0210] The following equipment was used: Agilent Technologies 1290 Inftnity UHPLC; Thermo Vanquish UHPLC; KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 shaker (Jintan Kexing Instrument Factory). Pure water system (Millipore); AS165W centrifuge (Asia Pacific (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); methanol (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Aladdin); ultrapure water; and all other reagents were analytical grade.

[0211] The standard decoction of Lithospermum erythrorhizon (Xinjiang Lithospermum erythrorhizon) and the standard decoction of counterfeit Lithospermum erythrorhizon (hard Lithospermum erythrorhizon, Potentilla chinensis) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0212] 2. Determination of chromatographic conditions:

[0213] The column was packed with octadecylsilane-bonded silica gel (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in the table below; the flow rate was 0.35 ml per minute; the column temperature was 40 °C; and the detection wavelength was 270 nm.

[0214]

[0215]

[0216] 3. Preparation of the test solution:

[0217] Take about 0.2g of sample powder, place it in a stoppered conical flask, add 25ml of water, seal tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, add 2% hydrochloric acid solution to adjust the pH to 1-2, extract twice with ethyl acetate, 25mL each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 3ml of methanol, and the sample is ready.

[0218] 4. Sample testing and data analysis:

[0219] Characteristic chromatograms of standard decoctions of Xinjiang Lithospermum and its adulterants (Lithospermum erythrorhizon and Potentilla chinensis) were determined and compared with those of the standard decoctions using the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission.

[0220] The results are as follows Figure 19As shown, peaks 4 and 10 are common characteristic peaks of Xinjiang Lithospermum and its adulterants (Potentilla chinensis and Lithospermum erythrorhizon). Peaks A, B, C, D, and E are unique peaks of Potentilla chinensis, and peak a is a unique peak of Lithospermum erythrorhizon. Therefore, the presence or absence of characteristic peaks can effectively distinguish genuine and adulterated Lithospermum. Furthermore, considering that peak 11 is a unique peak of Xinjiang Lithospermum, this characteristic peak can also be used to distinguish genuine and adulterated Lithospermum.

Claims

1. A method for constructing a UPLC feature map of Lithospermum erythrorhizon, characterized in that: Includes the following steps: 1) Preparation of reference solution for reference medicinal material: Take Xinjiang Lithospermum erythrorhizon reference medicinal material, add water, heat and extract, filter, treat the filtrate with hydrochloric acid solution, add ethyl acetate and shake to extract, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol to obtain the solution; 2) Preparation of reference solution: Take the set amount of protocatechuic acid reference standard, protocatechuic aldehyde reference standard, ferulic acid reference standard, rosmarinic acid reference standard, caffeic acid tetramer reference standard, and tanshinone reference standard, add methanol to prepare a mixed solution, which is used as the reference solution. 3) Preparation of test solution: Take a sample of Lithospermum erythrorhizon, which may be Lithospermum erythrorhizon herbal material, Lithospermum erythrorhizon slices or Lithospermum erythrorhizon standard decoction, extract with water, filter, treat the filtrate with hydrochloric acid solution, then extract with ethyl acetate by shaking, evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol to obtain the test solution; 4) Determination: Reference solutions of Xinjiang Lithospermum erythrorhizon, reference standard, and test sample were injected into a liquid chromatograph for determination, yielding characteristic chromatograms with 12 characteristic peaks. The mobile phase A of the liquid chromatograph was acetonitrile, and the mobile phase B was a 0.05%–0.2% (v / v) aqueous solution of phosphoric acid. The chromatographic column used was a CORTECS T3 with dimensions of 2.1 × 150 mm and a particle size of 1.6 μm. Gradient elution was employed: 0–20 min, 4% A → 15% A; 20 min–30 min, 15% A → 20% A; 30 min–33 min, 20% A. The detection wavelength was 270 nm, and an ultraviolet detector was used.

2. The method for constructing the UPLC feature map of Lithospermum erythrorhizon according to claim 1, characterized in that: In step 1), the volume of water added to the reference solution of Xinjiang Lithospermum erythrorhizon is 25ml to 100ml, the extraction time is 30min to 120min, the volume concentration of hydrochloric acid solution is 0.5% to 10%, the pH of the solution after treatment is 1 to 2, and the extraction with ethyl acetate is performed 1 to 3 times, with an extraction volume of 20ml to 100ml each time.

3. The method for constructing the UPLC feature map of Lithospermum erythrorhizon according to claim 1, characterized in that: In step 3), the volume of water added is 15ml to 50ml, the extraction time is 15min to 60min, the volume concentration of hydrochloric acid solution added is 0.5% to 10%, and the pH of the solution after treatment is 1 to 2; the number of extractions with ethyl acetate is 1 to 3, and the extraction volume each time is 20ml to 100ml.

4. The method for constructing the UPLC feature map of Lithospermum erythrorhizon according to claim 1, characterized in that: The flow rate measured in step 4) is 0.30 ml to 0.40 ml per minute; the column temperature is 35°C to 43°C.

5. The method for constructing the UPLC feature map of Lithospermum erythrorhizon according to claim 1, characterized in that: In step 4), the ultra-high performance liquid chromatography (UHPLC) chromatogram of the Xinjiang Lithospermum sample has 12 characteristic peaks, which should correspond to the retention times of the 12 characteristic peaks in the chromatogram of the reference medicinal material; among them, peak 1, peak 2, peak 4, peak 6, peak 11, and peak 12 should correspond to the retention times of the corresponding reference standard peaks.

6. The application of the method for constructing the UPLC characteristic map of Lithospermum erythrorhizon according to any one of claims 1-5 in distinguishing Lithospermum erythrorhizon samples from different origins; wherein the different origins are Lithospermum erythrorhizon from Xinjiang and Lithospermum erythrorhizon from Inner Mongolia.

7. The application of the method for constructing the UPLC feature map of Lithospermum according to any one of claims 1-5 in distinguishing Lithospermum and its counterfeit samples; wherein the counterfeit samples are Potentilla chinensis and Lithospermum erythrorhizon.

8. The method for constructing the UPLC characteristic spectrum of Lithospermum erythrorhizon according to any one of claims 1-5, applied to Lithospermum erythrorhizon medicinal materials, Lithospermum erythrorhizon slices, or Lithospermum erythrorhizon standard decoction.

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