Construction method and application of lithospermum erythrorhizon UPLC specific chromatogram

Abstract

The invention discloses a construction method and application of a lithospermum erythrorhizon UPLC (Ultra Performance Liquid Chromatography) specific chromatogram. The construction method comprises the following steps: 1) preparing a reference substance solution of a reference medicinal material; 2) preparation of a reference substance solution: taking a protocatechuic acid reference substance, a protocatechuic aldehyde reference substance, a ferulic acid reference substance, a rosmarinic acid reference substance, a caffeic acid tetramer reference substance and a tanshinol reference substance with set amounts, and adding methanol to prepare a mixed solution as the reference substance solution of the reference substance; (3) preparing a test solution; 4) determination: respectively sucking the reference substance solution of the reference material of the Sinkiang lithospermum, the reference substance solution of the reference substance and the solution of the test article, and injecting the solutions into a liquid chromatograph for determination to obtain a characteristic spectrum which comprises at least 12 characteristic peaks; and 5) carrying out qualitative analysis on the sample to be detected according to the characteristic chromatogram obtained in the above step. The specific chromatogram provided by the invention has 12 characteristic peaks, can effectively distinguish lithospermum (Sinkiang lithospermum) and counterfeit products thereof with different bases, and embodies the integrity of quality control of lithospermum.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine quality analysis and detection, and particularly relates to a method for constructing a UPLC characteristic fingerprint of Arnebia euchroma (Royle) Johnst. and its application. Background Art

[0002] The commercial Arnebia euchroma (Royle) Johnst. is the dried root of the plant Arnebia euchroma (Royle) Johnst. of the Boraginaceae family. It is dug in spring and autumn, removing sediment and dried. Arnebia euchroma (Royle) Johnst. was first recorded in "Shennong Ben Cao Jing", with a bitter taste and cold nature. It mainly treats pathogenic factors in the heart and abdomen, five types of jaundice, supplements middle qi, benefits the nine orifices, and promotes the flow of water channels. "Flora of China" records that Arnebia euchroma (Royle) Johnst. (soft Arnebia root) is mainly produced in Xinjiang and Tibet in China. It usually grows on gravelly sunny slopes, alluvial fans, grasslands, and meadows in alpine mountains at an altitude of 2100 - 3000m. It is the best-quality variety among Arnebia euchroma (Royle) Johnst. medicinal materials and has currently become the main source of commercial Arnebia euchroma (Royle) Johnst.

[0003] Arnebia euchroma (Royle) Johnst. mainly contains chemical components such as naphthoquinones, flavonoids, steroids, phenolic acids, alkaloids, and polysaccharides (Qian Xue, Li Haitao, Zeng Wanxiang, et al. Research progress on chemical components, pharmacological effects, and product applications of Arnebia euchroma (Royle) Johnst. [J]. Chinese Wild Plant Resources, 2021, 40(3): 52 - 56). Modern pharmacological research shows that it has the effects of clearing heat and cooling blood, promoting blood circulation to remove toxins, and promoting eruption and resolving macules, and can be used to treat various pharmacological activities such as blood heat toxin excess, purple macules, measles not erupting, sores, eczema, and scalds.

[0004] Huang Rui et al. used UPLC chromatography to determine the content of naphthoquinone components in wild and cultivated Arnebia euchroma (Royle) Johnst.; at the same time, two chemometric pattern recognition techniques, PCA and OPLS-DA, were used to determine that isobutyrylshikonin and β,β'-dimethylacryloylakankannin could distinguish wild and cultivated Arnebia euchroma (Royle) Johnst. (Huang Rui, Dai Shengyun, Wu Dongxue, et al. Comparative study on the quality of wild and cultivated Arnebia euchroma (Royle) Johnst. [J]. Chinese Journal of Pharmaceutical Analysis, 2024, 44(5): 783 - 795.). Hu Hezhula et al. analyzed and predicted the Q-Markers of Arnebia euchroma (Royle) Johnst., and naphthoquinones and polysaccharides could be used as candidate components for the total classification Q-Markers of Arnebia euchroma (Royle) Johnst. (Hu Hezhula, Alatan Chaolumen, Liao Cuiping, et al. Research progress and prediction of quality markers of Arnebia euchroma (Royle) Johnst. [J]. Acta Chinese Medicine and Pharmacology, 2024, 52(6): 105 - 110.). Roots of various medicinal plants in the Boraginaceae family containing naphthoquinone purple components are mixed and used as Arnebia euchroma (Royle) Johnst., and many imported Arnebia euchroma (Royle) Johnst. with unknown origins flow into the market, resulting in uneven quality of Arnebia euchroma (Royle) Johnst., seriously affecting the quality and efficacy of products. Existing literature mainly focuses on the study of liposoluble naphthoquinone components, with less research on water-soluble components, lacking an overall quality control method, and only some literature has conducted fingerprint analysis on Arnebia euchroma (Royle) Johnst. medicinal materials.

[0005] Ding Wenhuan et al. conducted a property and HPLC fingerprint analysis of Xinjiang Lithospermum officinale and yellow flower soft Lithospermum officinale, among which there were 5 common peaks, and the similarity was greater than 0.9. (Ding Wenhuan, Li Jie, Zhang Xuejia, et al. Establishment of HPLC fingerprint and chemical pattern recognition analysis of Xinjiang Lithospermum officinale and yellow flower soft Lithospermum officinale [J]. Chinese Patent Medicine, 2022, 44(10): 3220-3224.). Huang Rui et al. established an HPLC content determination and fingerprint analysis method for Lithospermum officinale materials, calibrated a total of 9 common peaks, and determined the content of 6 naphthoquinone chemical components. The results showed that the similarity of 50 batches of Lithospermum officinale materials varied greatly (Huang Rui, Dai Shengyun, Wu Dongxue, et al. Quality evaluation of Lithospermum officinale based on HPLC fingerprint and multi-index component content determination [J]. Chinese Journal of Pharmaceutical Sciences, 2024, 59(10): 929-937.). Through patent search, Minzu University of China disclosed a method and application of establishing a fingerprint spectrum of Xinjiang Lithospermum based on HPCE, and the capillary electrophoresis spectrum includes 6 characteristic peaks for distinguishing Xinjiang Lithospermum from Inner Mongolia Lithospermum (Minzu University of China. Method and application of establishing a fingerprint spectrum of Xinjiang Lithospermum based on HPCE: CN201710007836.8[P].2020-08-04.). The fingerprint spectrum established in the above document has a small number of characteristic peaks, and cannot simultaneously identify different origins of Lithospermum and authenticity, and cannot fully reflect the quality level of Xinjiang Lithospermum.

[0006] Therefore, it is necessary to develop new technologies in a targeted manner to effectively distinguish between lithospermum of different origins and their counterfeits, and to conduct overall control of the quality of lithospermum products. Summary of the invention

[0007] Purpose of the invention: In view of the shortcomings and defects of the prior art, the present invention provides a method for constructing a UPLC characteristic spectrum of Lithospermum officinale and its application. The characteristic spectrum has 12 characteristic peaks in total, which can effectively distinguish Lithospermum officinale (Xinjiang Lithospermum officinale) of different origins and its counterfeits, reflecting the integrity of Lithospermum officinale quality control, especially the overall evaluation and control of the quality of intermediates and finished products in each link of the production process of the formulated granule finished product.

[0008] Technical solution: A method for constructing a UPLC characteristic spectrum of Lithospermum officinale of the present invention is characterized by comprising the following steps:

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

[0010] 2) Preparation of the reference substance solution: Weigh a set amount of protocatechuic acid reference substance, protocatechualdehyde reference substance, ferulic acid reference substance, rosmarinic acid reference substance, caffeic acid tetramer reference substance, and danshensu reference substance, and dissolve them in methanol to prepare a mixed solution as the reference substance solution.

[0011] 3) Preparation of the test sample solution: Take the lithospermum erythrorhizon sample, extract it with water, filter, treat the filtrate with hydrochloric acid solution, then shake and extract with ethyl acetate. Evaporate the ethyl acetate solution to dryness, dissolve the residue in methanol to obtain the solution.

[0012] 4) Determination: Respectively pipette the reference substance solution of the lithospermum erythrorhizon reference crude drug, the reference substance solution, and the test sample solution, inject them into the liquid chromatograph for determination to obtain the characteristic chromatogram, and the characteristic chromatogram includes at least 12 characteristic peaks.

[0013] 5) Qualitative analysis of the test sample is carried out through the characteristic chromatogram obtained in the above steps.

[0014] Among them, in step 1) for the preparation of the reference substance solution of the lithospermum erythrorhizon reference crude drug, the volume of water added is 25 ml to 100 ml, the extraction time is 30 min to 120 min; the volume concentration of the hydrochloric acid solution added is 0.5% to 10%, and the pH of the treated solution is 1 to 2; the number of extractions with ethyl acetate is 1 to 3 times, and the volume of each extraction is 20 ml to 100 ml.

[0015] Among them, in step 2), the methanol solution is prepared by adding methanol to form an aqueous methanol solution with a volume concentration of 5% to 70%.

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

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

[0018] Among them, in step 4), the detection wavelength in the determination is 254 nm to 300 nm; the flow rate is 0.30 ml to 0.40 ml per minute; the column temperature is 35 °C to 43 °C; an ultraviolet detector is used.

[0019] Among them, the ultra-high performance liquid chromatography characteristic chromatogram of the Arnebia euchroma (Royle) Johnst. sample in step 4) has 12 characteristic peaks, and should correspond to the retention times of the 12 characteristic peaks in the reference chromatogram of the control medicinal material; among them, peak 1, peak 2, peak 4, peak 6, peak 11, and peak 12 should respectively correspond to the retention times of the corresponding reference substance peaks of the reference substances. The peak corresponding to the reference substance peak of 4-hydroxybenzoic acid is peak S1, and calculate the relative retention times of peak 3 and peak 5 with respect to peak S1; the peak corresponding to the reference substance of caffeic acid tetramer (Rabdosiin) is peak S2, and calculate the relative retention times of peak 7, peak 8, peak 9, and peak 10 with respect to peak S2, and their relative retention times should all be within the range of ±10% of the specified values. The specified values are: 0.92 (peak 3), 2.25 (peak 5), 0.74 (peak 7), 0.88 (peak 8), 0.90 (peak 9), 0.98 (peak 10).

[0020] Application of the method for constructing the UPLC characteristic chromatogram of Arnebia euchroma (Royle) Johnst. of the present invention in differentiating different original Arnebia euchroma (Royle) Johnst. samples.

[0021] Application of the construction method of the present invention in differentiating Arnebia euchroma (Royle) Johnst. and its counterfeit samples.

[0022] Application of the method for constructing the UPLC characteristic chromatogram of Arnebia euchroma (Royle) Johnst. of the present invention in Arnebia euchroma (Royle) Johnst. medicinal materials, Arnebia euchroma (Royle) Johnst. decoction pieces, Arnebia euchroma (Royle) Johnst. standard decoctions, Arnebia euchroma (Royle) Johnst. preparation intermediates or Arnebia euchroma (Royle) Johnst. preparation finished products.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention provides a characteristic chromatogram method that is simple to operate and can accurately distinguish different original Arnebia euchroma (Royle) Johnst. (Arnebia euchroma (Royle) Johnst.) and counterfeits. The characteristic chromatogram has a total of 12 characteristic peaks, which can effectively distinguish Arnebia euchroma (Royle) Johnst. (Arnebia euchroma (Royle) Johnst.) of different origins and their counterfeits, reflecting the integrity of the quality control of Arnebia euchroma (Royle) Johnst., especially the overall evaluation and control of the quality of intermediates and finished products in each link during the production process of the finished formula granules. To determine the quality stability of the finished product, better guide the production, and ensure the quality and efficacy of the product.

[0024] The method of the present invention has a short detection time, is simple to operate, has a small solvent consumption, and has less environmental pollution; after methodological verification, it has good reproducibility and stability, reliable recovery rates, low detection costs, and high detection efficiency. The present invention provides a new analytical means for the quality control of Arnebia euchroma (Royle) Johnst. (Arnebia euchroma (Royle) Johnst.), and at the same time can distinguish different origins and their counterfeits, providing an important guarantee for standardizing the production of Arnebia euchroma (Royle) Johnst. (Arnebia euchroma (Royle) Johnst.) preparations. The characteristic chromatogram of Arnebia euchroma (Royle) Johnst. established by the present invention has 12 characteristic peaks, and the presented chromatographic peak information is rich, containing a variety of chemical components, and can comprehensively control the quality of Arnebia euchroma (Royle) Johnst. (Arnebia euchroma (Royle) Johnst.) formula granules. Description of the Drawings

[0025] Figure 1It is the characteristic chromatogram of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules; Peak 1 in the figure: Danshensu; Peak 2: Protocatechuic acid; Peak 3: Protocatechualdehyde; Peak 4 (S1): 4-Hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Caffeic acid tetramer (Rabdosiin); Peak 12: Rosmarinic acid;

[0026] Figure 2 It is the characteristic chromatogram of the intermediate of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.); Peak 1 in the figure: Danshensu; Peak 2: Protocatechuic acid; Peak 3: Protocatechualdehyde; Peak 4 (S1): 4-Hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Caffeic acid tetramer (Rabdosiin); Peak 12: Rosmarinic acid;

[0027] Figure 3 It is the characteristic chromatogram of the standard decoction of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.); Peak 1 in the figure: Danshensu; Peak 2: Protocatechuic acid; Peak 3: Protocatechualdehyde; Peak 4 (S1): 4-Hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Caffeic acid tetramer (Rabdosiin); Peak 12: Rosmarinic acid;

[0028] Figure 4 It is the characteristic chromatogram of the cut crude drugs of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.); Peak 1 in the figure: Danshensu; Peak 2: Protocatechuic acid; Peak 3: Protocatechualdehyde; Peak 4 (S1): 4-Hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Caffeic acid tetramer (Rabdosiin); Peak 12: Rosmarinic acid;

[0029] Figure 5 It is the characteristic chromatogram of the medicinal materials of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.); Peak 1 in the figure: Danshensu; Peak 2: Protocatechuic acid; Peak 3: Protocatechualdehyde; Peak 4 (S1): 4-Hydroxybenzoic acid; Peak 6: Ferulic acid; Peak 11 (S2): Caffeic acid tetramer (Rabdosiin); Peak 12: Rosmarinic acid;

[0030] Figure 6 It is the chromatogram of reference substances, reference medicinal materials and Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules;

[0031] Figure 7 It is the UPLC chromatogram of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules at different detection wavelengths;

[0032] Figure 8 It is the UPLC diagram for investigating different extraction solvents;

[0033] Figure 9 It is the UPLC chromatogram for pH investigation;

[0034] Figure 10 It is the UPLC diagram for investigating different extraction methods;

[0035] Figure 11 It is the UPLC diagram for investigating different extraction volumes;

[0036] Figure 12 UPLC chromatograms examined at different extraction times;

[0037] Figure 13 UPLC chromatograms examined at different extraction times;

[0038] Figure 14 UPLC chromatograms examined with different extraction solvents;

[0039] Figure 15 Chromatogram of the column for the characteristic fingerprint of Lithospermum formula granules;

[0040] Figure 16 Chromatogram of the column temperature for the characteristic fingerprint of Lithospermum formula granules;

[0041] Figure 17 Chromatogram of the flow rate for the characteristic fingerprint of Lithospermum formula granules;

[0042] Figure 18 Comparison chart of the characteristic fingerprints of the standard decoctions of different original plants of Lithospermum;

[0043] Figure 19 Comparison chart of the characteristic fingerprints of the standard decoctions of genuine and fake Lithospermum;

[0044] Figure 20 Specificity test chart of Lithospermum formula granules;

[0045] Figure 21 Integrity test chart of Lithospermum formula granules. Detailed implementation manners

[0046] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and specific implementation manners.

[0047] The method for constructing the UPLC characteristic fingerprint of Lithospermum in the present invention includes the following steps:

[0048] 1) Select Lithospermum samples and prepare test solution; use Lithospermum (Arnebia euchroma) as the reference medicinal material to prepare the reference solution of the reference medicinal material; use danshensu, protocatechuic acid, protocatechualdehyde, 4-hydroxybenzoic acid, ferulic acid, tetramer of caffeic acid (Rabdosiin), rosmarinic acid as reference substances for reference standards, and prepare the reference solutions of reference standards respectively.

[0049] Preparation of reference solution: Take 0.5 g - 2 g of the reference crude drug of Arnebia euchroma (Royle) Johnst. (Arnebia guttata Bunge), place it in a stoppered conical flask, add 25 ml - 100 ml of water, heat under reflux for 30 minutes - 120 minutes, cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 0.5% - 10% hydrochloric acid solution, extract with ethyl acetate by shaking for 1 - 3 times, 20 ml - 100 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in methanol to obtain the reference crude drug reference solution. Preparation of reference substance solution: Take appropriate amounts of protocatechuic acid reference substance, 4-hydroxybenzoic acid reference substance, ferulic acid reference substance, and rosmarinic acid reference substance, weigh accurately, and prepare solutions containing 15 - 40 μg of protocatechuic acid reference substance, 4-hydroxybenzoic acid reference substance, ferulic acid reference substance, and rosmarinic acid per 1 ml in methanol respectively, and a solution containing 20 - 80 μg of sodium danshensu (equivalent to 18 - 72 μg of danshensu per 1 ml) as the reference substance solution.

[0050] Preparation of test solution: Take 0.2 g - 1.0 g of the Arnebia euchroma (Royle) Johnst. (Arnebia guttata Bunge) formula granules, grind them finely, place them in a stoppered conical flask, add 15 ml - 50 ml of water, tightly stopper, ultrasonically treat (power 250 W, frequency 40 kHz) for 15 minutes - 60 minutes, cool, shake well, filter, adjust the pH value of the subsequent filtrate to 1 - 2 with 0.5% - 10% hydrochloric acid solution, then extract with ethyl acetate by shaking for 1 - 3 times, 20 mL - 100 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in methanol to obtain the solution.

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

[0052] 2) Respectively absorb the reference solution and the test solution for ultra-high performance liquid chromatography analysis, construct the characteristic chromatogram of the sample to be tested, as Figure 1 shown, the characteristic chromatogram of the Arnebia euchroma (Royle) Johnst. (Arnebia guttata Bunge) formula granules includes at least 12 characteristic peaks, and the reference peaks in the characteristic peaks respectively correspond to danshensu, protocatechuic acid, protocatechualdehyde, 4-hydroxybenzoic acid, ferulic acid, tetramer of caffeic acid (Rabdosiin), and rosmarinic acid reference substances.

[0053] Carry out ultra-high performance liquid chromatography analysis on the test solution and the reference solution prepared in step 1) respectively to obtain the corresponding ultra-high performance liquid chromatograms; respectively accurately absorb 0.5 μl - 2 μl of the reference solution and the test solution, inject them into the liquid chromatograph, and measure to obtain.

[0054] Chromatographic condition parameters: The chromatographic column is packed with octadecylsilane chemically bonded silica; acetonitrile is used as mobile phase A, and 0.05% - 0.2% phosphoric acid solution is used as mobile phase B, and gradient elution is adopted; the flow rate is 0.30 ml - 0.40 ml per minute; the column temperature is 35°C - 43°C; an ultraviolet detector is used; the detection wavelength is 254 - 300 nm.

[0055] Preferably, octadecylsilyl silica gel is used as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 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 carried out according to the regulations in the following table; the flow rate is 0.35 ml per minute; the column temperature is 40 °C; the detection wavelength is 270 nm.

[0056]

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

[0058] Select the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) intermediate as the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) sample, repeat step 1), and the characteristic chromatogram of the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) intermediate constructed is as Figure 2 shown.

[0059] Select the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) standard decoction as the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) sample, repeat step 1), and the characteristic chromatogram of the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) standard decoction constructed is as Figure 3 shown

[0060] Select the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) medicinal materials as the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) sample, repeat step 1), and the characteristic chromatogram of the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) medicinal materials constructed is as Figure 4 shown.

[0061] Select the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) decoction pieces as the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) sample, repeat step 1), and the characteristic chromatogram of the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) decoction pieces constructed is as Figure 5 shown.

[0062] Such as Figure 6 , for the characteristic chromatogram of the Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules, 12 characteristic peaks should be presented in the test sample chromatogram, and the retention times of the 12 characteristic peaks should correspond to those in the reference chromatogram of the control medicinal materials; among them, peak 1, peak 2, peak 4, peak 6, peak 11, and peak 12 should respectively correspond to the retention times of the corresponding reference peaks of the reference substances. The peak corresponding to the 4-hydroxybenzoic acid reference substance is peak S1, and calculate the relative retention times of peak 3 and peak 5 with respect to peak S1; the peak corresponding to the rabdosiin reference substance is peak S2, and calculate the relative retention times of peak 7, peak 8, peak 9, and peak 10 with respect to peak S2, and the relative retention times should be within the range of ±10% of the specified values, and the specified values are: 0.92 (peak 3), 2.25 (peak 5), 0.74 (peak 7), 0.88 (peak 8), 0.90 (peak 9), 0.98 (peak 10).

[0063] 3) Based on the characteristic chromatogram obtained in step 2), perform the identification of the origin of the sample to be tested; the comparison of the control characteristic chromatograms of Arnebia euchroma (Royle) Johnst. and Arnebia guttata Bunge is as Figure 18 shown. Peaks 6, 7, and 11 in Arnebia euchroma (Royle) Johnst. were not detected in Arnebia guttata Bunge, and there is a specific peak A in Arnebia guttata Bunge.

[0064] 4) Based on the characteristic chromatogram obtained in step 2), perform the identification of genuine and fake samples of the sample to be tested. The comparison of the control characteristic chromatograms of Arnebia euchroma (Royle) Johnst. (Arnebia euchroma), fake Arnebia euchroma (Arnebia lithospermifolia Bunge, Potentilla chinensis Ser.) is as Figure 19 shown. There are significant differences between Arnebia euchroma (Royle) Johnst. and the fake samples. Only peaks 4 and 10 in Arnebia euchroma (Royle) Johnst. were detected in Arnebia lithospermifolia Bunge, and the specific peak is peak a. Peaks 2, 4, 10, and 12 in Arnebia euchroma (Royle) Johnst. were detected in Potentilla chinensis Ser., and the specific peaks are peaks A, B, C, D, and E.

[0065] The method for detecting the characteristic chromatogram of the Arnebia euchroma (Royle) Johnst. sample provided by the present invention can simultaneously perform the identification of different origins and genuine and fake samples. This method is convenient, fast, highly specific, and has good stability. Quality control is carried out from multiple aspects such as characteristic chromatogram, origin, and identification of genuine and fake samples, providing a more powerful guarantee for the industrialization of Arnebia euchroma (Royle) Johnst. formula granules.

[0066] Example 1:

[0067] Detection of Arnebia euchroma (Royle) Johnst. (Arnebia euchroma) formula granules, standard decoctions, and herbs / slices, and establishment of control chromatograms:

[0068] Take 3 batches of Arnebia euchroma (Royle) Johnst. (Arnebia euchroma) formula granules, 9 batches of Arnebia euchroma standard decoctions, and 9 batches of Arnebia euchroma herbs / slices as Arnebia euchroma samples, and perform quality control determinations according to the following steps respectively:

[0069] 1) Select Arnebia euchroma (Royle) Johnst. formula granules and prepare a test solution; use Arnebia euchroma as the control herb to prepare a reference solution of the control herb; use danshensu, protocatechuic acid, protocatechualdehyde, 4-hydroxybenzoic acid, ferulic acid, tetramer of caffeic acid (Rabdosiin), and rosmarinic acid as reference substances for reference solutions, and prepare reference solutions of reference substances respectively.

[0070] Preparation of the reference solution: Take 1 g of the control herb of Arnebia euchroma, place it in a stoppered conical flask, add 50 ml of water, heat under reflux for 60 minutes, cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 50 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the reference solution of the control herb.

[0071] Preparation of reference substance solution: Weigh appropriate amounts of reference substances of danshensu, protocatechuic acid, protocatechuic aldehyde, 4-hydroxybenzoic acid, ferulic acid, tetramer of caffeic acid (Rabdosiin), and rosmarinic acid accurately, and dissolve them in 10% methanol to prepare a mixed solution containing 20 μg of protocatechuic acid reference substance, 4-hydroxybenzoic acid reference substance, ferulic acid reference substance, and rosmarinic acid reference substance per 1 ml, and 40 μg of sodium danshensu reference substance (equivalent to 36 μg of danshensu per 1 ml) per 1 ml as the reference substance solution.

[0072] Preparation of test solution for Zicao formula granules: Take about 0.5 g of Zicao formula granules, place them in a stoppered conical flask, add 25 ml of water, stopper tightly, and ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 25 ml each time. Combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the solution.

[0073] Preparation of test solution for Zicao standard decoction: Take about 0.2 g of the standard decoction powder, place it in a stoppered conical flask, add 25 ml of water, stopper tightly, and ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 25 ml each time. Combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the solution.

[0074] Preparation of test solution for Zicao medicinal materials / slices: Take 1 g of Zicao medicinal materials / slices, place them in a stoppered conical flask, add 50 ml of water, heat under reflux for 60 minutes, let it cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 50 ml each time. Combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the solution.

[0075] 2) Perform ultra-high performance liquid chromatography analysis on the test solution and the reference substance solution prepared in step 1) respectively to obtain the corresponding ultra-high performance liquid chromatography diagrams; accurately pipette 1 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph for determination to obtain the results.

[0076] Among them, the chromatographic condition parameters are as follows:

[0077] Using octadecylsilane chemically bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; the flow rate is 0.35 ml per minute; the column temperature is 40 °C; the detection wavelength is 270 nm. The number of theoretical plates calculated based on the peak of tetramer of caffeic acid (Rabdosiin) should be not less than 5000.

[0078]

[0079] As shown Figure 6 in the figure, for the characteristic chromatogram of Lithospermum erythrorhizon formula granules, 12 characteristic peaks should appear in the chromatogram of the test sample, and the retention times of the 12 characteristic peaks should correspond to those in the chromatogram of the reference medicinal material; among them, peak 1, peak 2, peak 4, peak 6, peak 11, and peak 12 should respectively correspond to the retention times of the corresponding reference peaks of the reference substances. The peak corresponding to the reference peak of 4-hydroxybenzoic acid reference substance is peak S1, and calculate the relative retention times of peak 3 and peak 5 with respect to peak S1; the peak corresponding to the reference substance of caffeic acid tetramer (Rabdosiin) reference substance is peak S2, and calculate the relative retention times of peak 7, peak 8, peak 9, and peak 10 with respect to peak S2, and their relative retention times should all be within the range of ±10% of the specified values. The specified values are: 0.92 (peak 3), 2.25 (peak 5), 0.74 (peak 7), 0.88 (peak 8), 0.90 (peak 9), 0.98 (peak 10). The determination results are shown in Tables 1, 2, and 3 below.

[0080] Table 1 Determination Results of Lithospermum erythrorhizon (Arnebia euchroma) Formula Granule Samples (Relative Retention Time)

[0081]

[0082] Table 2 Determination Results of Lithospermum erythrorhizon (Arnebia euchroma) Medicinal Material / Slice Samples (Relative Retention Time)

[0083]

[0084] Table 3 Determination Results of Lithospermum erythrorhizon (Arnebia euchroma) Standard Decoction Samples (Relative Retention Time)

[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] From the above test results, it can be seen analytically that the characteristic chromatograms of 3 batches of Lithospermum erythrorhizon formula granules, 9 batches of Lithospermum erythrorhizon standard decoctions, and medicinal materials / slices all meet the requirements of the characteristic chromatogram in step 2).

[0088] This example is used to confirm the optimal scheme for establishing the characteristic chromatogram of Lithospermum erythrorhizon samples, specifically including the comparison of chromatographic conditions, wavelength selection, and preparation schemes of test solution samples, and select the optimal chromatographic conditions and test solution preparation schemes; and investigate the specificity, integrity, precision, intermediate precision, stability, repeatability, and durability of the optimal scheme respectively.

[0089] Example 2:

[0090] Establishment of the characteristic chromatogram method for Lithospermum officinale formula granules and its methodology research:

[0091] This example is used to confirm the optimal scheme for establishing the characteristic chromatogram in the quality control method of Lithospermum officinale formula granules, specifically including the comparison of chromatographic conditions, wavelength selection, and the preparation of test solution, and selecting the optimal chromatographic conditions and test solution preparation scheme; and the specificity, integrity, precision, intermediate precision, stability, repeatability, and durability of the optimal scheme are investigated respectively.

[0092] 1. Instruments and reagents:

[0093] Agilent Tcchnologies 1290Inftnity ultra-high performance liquid chromatograph; Thermo Vanquish ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); thermostatic water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 oscillator (Jintan Kexing Instrument Factory). Pure water system (Millipore); AS165W centrifuge (As One (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); methanol (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, aladdin); water is ultrapure water; other reagents are all of analytical grade.

[0094] Reference standards of sodium danshensu, protocatechuic acid, 4-hydroxybenzoic acid, ferulic acid, rosmarinic acid, and protocatechualdehyde were all purchased from the National Institutes for Food and Drug Control, and their batch numbers are 110855-201915, 110809-201906, 101149-202204, 110773-201915, 111871-201505, and 110810-201909 respectively.

[0095] Reference standard of rabdosiin was purchased from Chengdu Push Biotechnology Co., Ltd., and the batch number is PS013125.

[0096] Reference medicinal material of Lithospermum officinale (Arnebia euchroma) was purchased from the National Institutes for Food and Drug Control, and the batch number is 121430-201504.

[0097] Lithospermum officinale (Arnebia euchroma) formula granules, intermediates, medicinal materials / herbal pieces, and standard decoctions were all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0098] 2. Determination of detection wavelength:

[0099] Take about 0.5g of this product, put it in a stoppered conical flask, add 25ml of water, seal it, and treat it with ultrasound (power 250W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, add 2% hydrochloric acid solution to the filtrate to adjust the pH value to 1-2, shake it with ethyl acetate for 2 times, 25ml each time, combine the ethyl acetate liquid, evaporate it to dryness, and add 3ml of methanol to dissolve the residue to obtain it. Collect the chromatograms of the sample at 254nm, 270nm and 300nm, such as Figure 7 shown.

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

[0101] 3. Determination of chromatographic conditions:

[0102] Octadecylsilane bonded silica gel was used as the filler (column length was 150 mm, inner diameter was 2.1 mm, particle size was 1.6 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in the following table; 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 test solution:

[0105] 4.1. Investigation of different extraction solvents:

[0106] Take an appropriate amount of lithospermum officinale (Xinjiang lithospermum officinale) granules, grind them into powder, take about 0.5g, and make 5 parallel portions, accurately weigh them, put them in a stoppered conical flask, add water, 30% methanol, 50% methanol, 80% methanol, and 25ml methanol, respectively, plug them, and ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, add 2% hydrochloric acid solution to the filtrate to adjust the pH value to 1-2, shake and extract with ethyl acetate twice, 25mL each time, combine the ethyl acetate liquid, evaporate to dryness, and add 3ml of methanol to the residue to dissolve. Accurately draw 1μl of each test solution, inject it into the liquid chromatograph, measure it according to the above chromatographic conditions, calculate the peak area / sample weight of the characteristic peak, and the results are shown in Table 4. Figure 8 .

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

[0108]

[0109] The results show that when the extraction solvent is water, the extraction efficiency is higher, and water is determined to be the extraction solvent.

[0110] 4.2. pH investigation:

[0111] Take an appropriate amount of Lithospermum erythrorhizon (Arnebia euchroma) formula granules, grind them finely, take about 0.5 g, in duplicate for each of the 2 parallel groups, place them in a stoppered conical flask, add 25 ml of water, tightly stopper, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract twice with ethyl acetate by shaking, 25 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the solution. Precisely pipette 1 μl of each test solution respectively, inject it into the liquid chromatograph, and determine according to the above chromatographic conditions, calculate the peak area of the characteristic peak / sample weight, and the results are shown in Table 5. Figure 9 。

[0112] Table 5 Investigation of characteristic chromatogram pH (peak area / sample weight)

[0113]

[0114] The results show that: the response of the characteristic peak is higher when adjusting the pH, so it is determined that the preparation method of the test sample needs to adjust the pH.

[0115] 4.3. Investigation of different extraction methods:

[0116] Take an appropriate amount of Lithospermum erythrorhizon (Arnebia euchroma) formula granules, grind them finely, take about 0.5 g, in duplicate for each of the 2 parallel groups, place them in a stoppered conical flask, add 25 ml of water, tightly stopper, and ultrasonically treat, shake, and reflux for 30 minutes respectively, cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract twice with ethyl acetate by shaking, 25 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the solution. Precisely pipette 1 μl of each test solution respectively, inject it into the liquid chromatograph, and determine according to the above chromatographic conditions, calculate the peak area of the characteristic peak / sample weight, and the results are shown in Table 6. Figure 10 。

[0117] Table 6 Investigation of characteristic chromatogram extraction method (peak area / sample weight)

[0118]

[0119] The results show that: the extraction efficiencies of different extraction methods are not very different. Considering the simplicity of the experimental operation, ultrasonic treatment is selected as the extraction method.

[0120] 4.4. Investigation of different extraction volumes:

[0121] Take an appropriate amount of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules, grind them finely, take about 0.5 g, parallel 4 groups, with 2 parallels in each group. Place them in a stoppered conical flask, add 15 ml, 25 ml, and 50 ml of water respectively, stopper tightly, and perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 25 mL each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 3 ml of methanol. Accurately pipette 1 μl of each test solution respectively, inject it into the liquid chromatograph, and determine according to the above chromatographic conditions. 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 show that: the extraction efficiencies of different extraction volumes are not very different, and the response of the characteristic peak is larger when the extraction solvent is 25 ml. Therefore, 25 ml of extraction solvent is selected.

[0125] 4.5. Investigation of different extraction times:

[0126] Take an appropriate amount of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules, grind them finely, take about 0.5 g, parallel 4 portions, place them in a stoppered conical flask, add 25 ml of water, stopper tightly, and perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes. Let it cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 25 mL each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 3 ml of methanol to obtain. Accurately pipette 1 μl of each test solution respectively, inject it into the liquid chromatograph, and determine according to the above chromatographic conditions. 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 efficiencies at different extraction times (peak area / sample weight)

[0128]

[0129] The results show that: when the ultrasonic treatment is for 30 minutes, the extraction efficiency is relatively high. Considering the aspects of complete extraction and time saving, the extraction time is determined to be 30 minutes.

[0130] 4.6. Investigation of different extraction times:

[0131] Take an appropriate amount of Lithospermum erythrorhizon (Arnebia euchroma) formula granules, grind them finely, take about 0.5 g, parallel 4 groups, 2 portions in each group in parallel, place them in a stoppered conical flask, add 25 ml of water, tightly stopper, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 1 time, 2 times, and 3 times respectively, 25 mL each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain. Precisely pipette 1 μl of each test solution respectively, inject it into the liquid chromatograph, and determine according to the above chromatographic conditions, 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 show that: after extraction 2 times, the extraction efficiency tends to be stable. Considering comprehensively from the perspectives of sufficient extraction and time saving, it is determined to extract 2 times.

[0135] 4.7. Investigation of different extraction solvents:

[0136] Take an appropriate amount of Lithospermum erythrorhizon (Arnebia euchroma) formula granules, grind them finely, take about 0.5 g, parallel 4 groups, 2 portions in each group in parallel, place them in a stoppered conical flask, add 25 ml of water, tightly stopper, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate and n - butanol by shaking 2 times respectively, 25 mL each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain. Precisely pipette 1 μl of each test solution respectively, inject it into the liquid chromatograph, and determine according to the above chromatographic conditions, 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 show that: when the extraction solvent is ethyl acetate, the extraction efficiency is higher, and it is determined that the extraction solvent is ethyl acetate.

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

[0141] According to the above research results, the preparation method of the test solution for the characteristic chromatogram of Lithospermum erythrorhizon formula granules is determined as:

[0142] Take an appropriate amount of Lithospermi Radix (Arnebia euchroma) formula granules, grind them finely, take about 0.5 g, place it in a stoppered conical flask, add 25 ml of water, stopper tightly, perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 25 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the solution.

[0143] 5. Research on the methodology of characteristic chromatogram:

[0144] 5.1. Specificity investigation:

[0145] Inject the test solution of Lithospermi Radix (Arnebia euchroma) formula granules and the negative solution without Lithospermi Radix into the liquid chromatograph, and the results are as follows Figure 20 .

[0146] The experimental results show that there are no chromatographic peaks at the retention times of each characteristic peak in the chromatogram of the test solution for the negative solution, indicating that the solvent and excipients have no interference on the determination of Lithospermi Radix formula granules, and this method for determining Lithospermi Radix formula granules has specificity.

[0147] 5.2. Integrity investigation:

[0148] Under the determined chromatographic conditions, extend the elution time to investigate whether there will be residual impurity peaks affecting the subsequent samples in the established chromatographic condition system. The results are as follows Figure 21 .

[0149] It can be seen from the experimental results that when the elution time is extended, there are no impurity peaks, indicating that the chromatographic conditions basically meet the principle of maximum information content and have no influence on the analysis of subsequent samples.

[0150] 5.3. Precision investigation:

[0151] Take a batch of Lithospermi Radix (Arnebia euchroma) formula granules, prepare the test solution according to the preparation method of the test solution, inject continuously 6 times, 1 μl each time, record the retention times of its characteristic peaks, and calculate the relative retention time according to the requirements in the main text. The results are shown in Table 11.

[0152] Table 11 Results of precision experiment (relative retention time)

[0153]

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

[0155] 5.4. Intermediate precision investigation:

[0156] Take a batch of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules, prepare a test solution according to the preparation method of the test solution, and have two experimenters A and B respectively prepare 3 portions according to the preparation method of the test solution, inject 1 μl on Agilent and Waters instruments respectively, record the retention time of the characteristic peaks, and calculate the relative retention time according to the requirements of the main text. The results are shown in Table 12.

[0157] Table 12 Intermediate precision test (relative retention time)

[0158]

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

[0160] 5.5, Stability test:

[0161] Take a batch of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules, prepare a test solution according to the preparation method of the test solution, inject 1 μl at 0, 2, 4, 8, 12, 16, and 24 hours respectively, record the retention time of the characteristic peaks, 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 test (relative retention time)

[0163]

[0164] The results show that the RSD of the relative retention times of each characteristic peak is less than 1%, and the test solution is stable within 24 hours.

[0165] 5.6, Repeatability test:

[0166] Take a batch of Lithospermum erythrorhizon (Lithospermum erythrorhizon Sieb. et Zucc.) formula granules, prepare 6 parallel groups, prepare a test solution according to the preparation method of the test solution, inject 1 μl respectively, record the retention time of the characteristic peaks, and calculate the relative retention time according to the requirements. The results are shown in Table 14.

[0167] Table 14 Repeatability test (relative retention time)

[0168]

[0169]

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

[0171] 5.7, Robustness test:

[0172] 5.7.1, Chromatographic column test:

[0173] Take a batch of Lithospermum erythrorhizon (Arnebia euchroma) formula granules, prepare the test solution according to the preparation method of the test solution, and use Syncronis C18 (2.1 mm × 100 mm, 1.7 μm); GLOD aq (2.1 mm × 150 mm, 1.9 μm); CORTECS T3 (2.1 * 150 mm, 1.6 um) three chromatographic columns respectively. Inject 1 μl respectively and record the corresponding chromatograms. The results are shown in Table 15. Figure 15 。

[0174] Table 15 Investigation of chromatographic columns (relative retention time)

[0175]

[0176] The results show that there is a peak wrapping phenomenon in the Syncronis C18 (2.1 mm × 100 mm, 1.7 μm) chromatographic column. The relative retention times and relative peak areas of each characteristic peak measured by the GLOD aq (2.1 mm × 150 mm, 1.9 μm) and CORTECS T3 (2.1 * 150 mm, 1.6 um) chromatographic columns are similar, and the resolution of each characteristic peak is good, indicating good durability. Therefore, the recommended chromatographic column for the detection of the characteristic chromatogram of Lithospermum erythrorhizon formula granules is CORTECS T3 (2.1 * 150 mm, 1.6 um).

[0177] 5.7.2. Column temperature investigation:

[0178] Take a batch of Lithospermum erythrorhizon (Arnebia euchroma) formula granules, prepare the test solution according to the preparation method of the test solution, investigate three temperatures of 35 °C, 40 °C, and 43 °C, inject 1 μl respectively, and record the retention times of its characteristic peaks. The results are shown in Table 16. Figure 16 。

[0179] Table 16 Investigation of column temperature (relative retention time)

[0180]

[0181] The results show that the determination results of the samples are relatively stable in the range of 35 °C to 43 °C for the column temperature and all meet the retention time requirements specified in the method, indicating good durability.

[0182] 5.7.3. Flow rate investigation:

[0183] Take a batch of Lithospermum erythrorhizon (Arnebia euchroma) formula granules, prepare the test solution according to the preparation method of the test solution, investigate the separation effect of the samples at three flow rates of 0.3 ml per minute, 0.35 ml per minute, and 0.4 ml per minute. The results are shown in Table 17. Figure 17 。

[0184] Table 17 Investigation of flow rate (relative retention time)

[0185]

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

[0187] Example 3:

[0188] Identification of different origins of Arnebiae Radix:

[0189] 1. Instruments and reagents:

[0190] Agilent Tcchnologies 1290Inftnity ultra-high performance liquid chromatograph; Thermo Vanquish ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 oscillator (Jintan Keyuan Instrument Factory). Pure water system (Millipore); AS165W centrifuge (As One (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); methanol (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, aladdin); water was ultrapure water; other reagents were all of analytical grade.

[0191] The standard decoctions of Arnebiae Radix (Arnebia euchroma) and Arnebiae Radix (Arnebia guttata Bunge) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0192] 2. Determination of chromatographic conditions:

[0193] Using octadecylsilane chemically bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in the following table; the flow rate was 0.35 ml per minute; the column temperature was 40 °C; the detection wavelength was 270 nm.

[0194]

[0195] 3. Preparation of test solution:

[0196] Take about 0.2 g of the sample powder, place it in a stoppered conical flask, add 25 ml of water, tightly stopper, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 25 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the solution.

[0197] 4. Sample determination and data analysis:

[0198] Take 7 batches of standard decoctions of Arnebia euchroma from Xinjiang and 3 batches of standard decoctions of Arnebia guttata from Inner Mongolia respectively. Prepare sample test solutions according to the above method for preparing test solutions of the samples. Precisely pipette 1 μl and inject it into the liquid chromatograph. According to the above chromatographic conditions, determine the characteristic fingerprints of the standard decoctions of Arnebia euchroma from Xinjiang and Arnebia guttata from Inner Mongolia respectively. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)" recommended by the Pharmacopoeia Commission of the People's Republic of China to generate reference characteristic fingerprints, and then import the two reference characteristic fingerprints into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)" for comparative analysis.

[0199] The results are as Figure 18 shown. Some characteristic peaks in the standard decoction of Arnebia euchroma from Xinjiang were not detected in Arnebia guttata from Inner Mongolia, and only 9 common characteristic peaks in Arnebia euchroma were presented. Peaks 6, 7, and 11 were not detected in the standard decoction of Arnebia guttata from Inner Mongolia, and peak A was a unique peak in Arnebia guttata from Inner Mongolia.

[0200] Further analyze the differences between the standard decoctions of Arnebia euchroma from Xinjiang and Arnebia guttata from Inner Mongolia through relative peak area. Taking peak 4 as the reference peak, compare the relative peak area differences of each characteristic peak in the two standard decoctions. The results are shown in Tables 18 and 19.

[0201] Table 18 Relative peak areas of standard decoctions of Arnebia euchroma from Xinjiang and those of different original plants (peak 4: S peak)

[0202]

[0203]

[0204] Table 19 Differences in relative peak areas of standard decoctions of Arnebia euchroma (from Xinjiang) and Arnebia guttata (from Inner Mongolia)

[0205]

[0206] The results show that there is no overlap in the relative peak area ranges of peaks 9, 10, and 12 between Arnebia euchroma from Xinjiang and Arnebia guttata from Inner Mongolia, and the established characteristic method can effectively distinguish samples of Arnebia with different original plants.

[0207] Example 4:

[0208] Identification of genuine and fake Arnebia:

[0209] 1. Instruments and reagents:

[0210] Agilent Technologies 1290 Infinity ultra-high performance liquid chromatograph; Thermo Vanquish ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 oscillator (Jintan KeXing Instrument Factory). Pure water system (Millipore); AS165W centrifuge (ASONE (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); methanol (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, aladdin); water is ultrapure water; other reagents are of analytical grade.

[0211] The standard decoctions of Arnebia euchroma (Royle) Johnst. and its adulterants (Arnebia szechenyi Kanitz, Potentilla chinensis Ser.) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0212] 2. Determination of chromatographic conditions:

[0213] Using octadecylsilane chemically bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in the following table; the flow rate was 0.35 ml per minute; the column temperature was 40 °C; the detection wavelength was 270 nm.

[0214]

[0215]

[0216] 3. Preparation of test solution:

[0217] Take about 0.2 g of the sample powder, place it in a stoppered conical flask, add 25 ml of water, tightly stopper, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, adjust the pH value of the filtrate to 1 - 2 with 2% hydrochloric acid solution, extract with ethyl acetate by shaking 2 times, 25 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 3 ml of methanol to obtain the solution.

[0218] 4. Sample determination and data analysis:

[0219] The characteristic fingerprints of the standard decoctions of Arnebia euchroma (Royle) Johnst. and its adulterants (Arnebia szechenyi Kanitz, Potentilla chinensis Ser.) were determined respectively, and comparative analysis was carried out using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)" recommended by the Pharmacopoeia Commission of the People's Republic of China.

[0220] The results are as Figure 19As shown in the figure, peaks 4 and 10 are the common characteristic peaks of Arnebia euchroma (Royle) Johnst. and its counterfeits (Potentilla chinensis Ser. and Arnebia szechenyi Kanitz). Peaks A, B, C, D, and E are the specific peaks of Potentilla chinensis Ser., and peak a is the specific peak of Arnebia szechenyi Kanitz. Therefore, the authenticity of Arnebia euchroma (Royle) Johnst. can be effectively distinguished by the presence or absence of characteristic peaks. At the same time, considering that peak 11 is the specific peak of Arnebia euchroma (Royle) Johnst., the authenticity of Arnebia euchroma (Royle) Johnst. can be distinguished by this characteristic peak.

Claims

1. A method for constructing a UPLC characteristic spectrum of Lithospermum officinale, characterized in that: The steps include: 1) Preparation of reference medicinal material solution: Take Xinjiang Lithospermum officinale reference medicinal material, add water, heat and extract, filter, add hydrochloric acid solution to the filtrate, and then add ethyl acetate to shake and extract, evaporate the ethyl acetate solution to dryness, and dissolve the residue in methanol to obtain; 2) Preparation of reference substance solution: taking a set amount of protocatechuic acid reference substance, protocatechuic aldehyde reference substance, ferulic acid reference substance, rosmarinic acid reference substance, caffeic acid tetramer reference substance, and danshensu reference substance, adding methanol to prepare a mixed solution as the reference substance solution; 3) Preparation of test solution: Take a sample of Lithospermum officinale, extract it with water, filter it, treat the filtrate with hydrochloric acid solution, then add ethyl acetate to shake and extract, evaporate the ethyl acetate solution to dryness, and dissolve the residue with methanol to obtain the solution; 4) Determination: Take the reference solution of Xinjiang Lithospermum officinale, the reference solution of the reference substance and the test solution respectively, inject them into the liquid chromatograph for determination, and obtain the characteristic spectrum. Its characteristic spectrum includes at least 12 characteristic peaks; 5) Perform qualitative analysis of the sample to be tested using the characteristic spectrum obtained in the above steps.

2. The method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to claim 1, wherein: In the step 1), the volume of water added in the preparation of the Xinjiang Lithospermum officinale reference medicinal material reference solution is 25ml-100ml, and the extraction time is 30min-120min; the volume concentration of the hydrochloric acid solution added is 0.5%-10%, and the pH value of the solution after treatment is 1-2; the number of extractions with ethyl acetate is 1-3 times, and the volume of each extraction is 20ml-100ml.

3. The method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to claim 1, wherein: In the step 2), methanol is added to prepare a methanol aqueous solution with a volume concentration of 5% to 70%.

4. The method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to claim 1, wherein: In the step 3), the volume of water added is 15ml-50ml, and the extraction time is 15min-60min; the volume concentration of the hydrochloric acid solution added is 0.5%-10%, and the pH value of the solution after treatment is 1-2; the number of extractions with ethyl acetate is 1-3 times, and the volume of each extraction is 20ml-100ml.

5. The method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to claim 1, wherein: In the 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 in the determination is filled with octadecylsilane bonded silica gel; and the 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.

6. The method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to claim 5, characterized in that: In the step 4), the detection wavelength is 254nm-300nm; the flow rate is 0.30ml-0.40ml per minute; the column temperature is 35°C-43°C; and an ultraviolet detector is used.

7. The method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to claim 1, characterized in that: The ultra-high performance liquid chromatography characteristic spectrum of the Xinjiang Lithospermum officinale sample in the step 4) has 12 characteristic peaks, and the retention times of the 12 characteristic peaks in the chromatogram of the reference medicinal material reference should correspond to each other; wherein peak 1, peak 2, peak 4, peak 6, peak 11, and peak 12 should correspond to the retention times of the corresponding reference substance peaks, respectively.

8. The application of the method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to any one of claims 1-7 in distinguishing different original Lithospermum officinale samples.

9. Application of the method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to any one of claims 1 to 7 in distinguishing samples of Lithospermum officinale and its counterfeits.

10. Application of the method for constructing the UPLC characteristic spectrum of Lithospermum officinale according to any one of claims 1 to 7 in Lithospermum officinale medicinal materials, Lithospermum officinale decoction pieces, Lithospermum officinale standard decoction, Lithospermum officinale preparation intermediates or Lithospermum officinale preparation finished products.

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