Characteristic chromatogram construction method of Tianlong or preparation thereof and method for identifying Tianlong medicinal material adulterants

Abstract

The invention provides a construction method of a characteristic spectrum of teloon or a preparation thereof. The invention also provides a method for identifying the Tianlong medicinal material adulterants. The construction method of the characteristic chromatogram comprises the following steps: taking a Tianlong reference medicinal material and a pterixin-6-carboxylic acid reference substance as parameter substances, determining eight characteristic peaks, and identifying that the peak 3 is hypoxanthine, the peak 4 is thymine and the peak 5 is pterixin-6-carboxylic acid; the eight characteristic peaks stably exist in the gecko medicinal material, the decoction pieces, the standard decoction and the formula granules, which indicates that the gecko medicinal material, the decoction pieces, the standard decoction and the formula granules are consistent in material basis and are all from the gecko medicinal material, so that the conditions of the gecko and the preparation thereof can be effectively and comprehensively reflected, and the gecko can be distinguished from other easily-mixed products (such as gecko).

Description

Technical Field

[0001] This invention relates to a method for constructing a characteristic spectrum of Tianlong or its preparations, and a method for identifying adulterants of Tianlong medicinal materials. Background Technology

[0002] Tianlong refers to the dried whole body of the Japanese gecko (Gekko japonicas, Dumeril et Bibron), a species of gecko. It is listed in the 1994 edition of the *Shanghai Standards for Traditional Chinese Medicine* and possesses the effects of relieving wind and spasms, dispelling wind and relieving pain, and attacking toxins and dissipating nodules. It is used for infantile convulsions, epilepsy, tetanus, rheumatic pain, scrofula, and carbuncles. Quality testing information for Tianlong is mostly based on its physical characteristics.

[0003] Application No. 202111233786.8, Invention Title: Method for Detecting Characteristic Spectra of Tianlong Formula Granules, relates to a method for detecting characteristic spectra of Tianlong formula granules. The method uses high performance liquid chromatography for analysis, employing a chromatographic column packed with octadecylsilane-bonded silica gel, with methanol as mobile phase A and 0.15% phosphoric acid aqueous solution as mobile phase B for gradient elution, a mobile phase flow rate of 0.9 ml / min, a column temperature of 25-35℃, and a detection wavelength of 295 nm. This patent document has the following shortcomings: (1) the number of characteristic peaks is relatively small (6), and only one characteristic peak (pterin-6-carboxylic acid) is identified; (2) it only uses Tianlong formula granules as the research object, ignoring the premise that the formula granules originally came from medicinal materials, and does not use Tianlong reference medicinal materials as a reference. Whether this method can effectively characterize the characteristics of Tianlong preparations is debatable.

[0004] Application No. 202210627638.2, Invention Title: A Comprehensive Method for Controlling the Quality of Gecko Formula Granules, discloses a comprehensive method for controlling the quality of gecko formula granules. This method includes a thin-layer chromatography (TLC) identification method and a liquid chromatography (LC) method for simultaneously performing characteristic chromatographic analysis and determining the content of tyrosine and tryptophan. This invention establishes a rapid, comprehensive, and highly specific set of quality detection methods for gecko formula granules. By controlling quality from multiple perspectives—including TLC identification, characteristic chromatographic analysis, and content determination—it provides stronger support for the industrialization of gecko formula granules.

[0005] Bao Huayin, Research on Key Technologies and Quality Evaluation System for Quality Control of Gecko (a Chinese medicinal herb), Shandong University of Traditional Chinese Medicine, 2012, published a fingerprint chromatographic study of Gecko. The published detection conditions were: Thermo 0DS-2 HYPERSIL column (particle size 54 μm, 100 Å).

[0006] The column temperature was 25℃ (4.6mm x 250mm, Thermo Scientific), the mobile phase was acetonitrile-water, the flow rate was 1 mL / min, the injection volume was 20 L, and the detection wavelength was 205 nm. The chromatograms in these two articles exhibited baseline drift, peak broadening, and abnormal peak purity, making them unsuitable as fingerprint chromatograms for quality control. Geckos are a general term for animals in the family Gekkonidae of the order Squamata, with numerous species, currently known to number in the thousands. The gecko studied in the two articles is the webbed gecko *Gekko swinhoana* Güenther, which differs significantly from the warty gecko *Gekko japonicas* (Dumeril et Bibron) in terms of specific components and characteristic chromatograms compared to this study.

[0007] The difficulty in identifying Tianlong (Gecko multivar) lies mainly in its high similarity in appearance to closely related species and common counterfeits. In particular, its characteristics become blurred after drying, and it is even more difficult to distinguish genuine products from counterfeits after being powdered or extracted. Summary of the Invention

[0008] In response to the above situation, the present invention establishes a method for constructing the characteristic spectrum of Tianlong and its preparations.

[0009] This invention provides a method for constructing the characteristic spectrum of Tianlong or its preparations, which includes the following steps:

[0010] a. Preparation of the test solution:

[0011] Take the Tianlong sample, dissolve and extract it with a solvent to obtain the test solution;

[0012] b. The test solution was analyzed by high performance liquid chromatography to obtain the characteristic chromatogram of Tianlong or its preparations;

[0013] The high-performance liquid chromatography (HPLC) conditions are as follows: a C18 column; acetonitrile as mobile phase A; 0.1% acetic acid solution as mobile phase B; gradient elution; and the gradient elution conditions are as follows:

[0014]

[0015] The preparation method of the test sample solution is as follows: take 0.5g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of water, seal tightly, weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with water, shake it well, filter it, and take the filtrate to obtain the test sample solution; the sonication conditions are: power 600W, frequency 40kHz, and treatment time: 30 minutes.

[0016] The mobile phase flow rate is 1.0 ml per minute; the column temperature is 30°C; the detection wavelength is 260 nm; and the theoretical plate number, calculated based on the pterin-6-carboxylic acid peak, should be no less than 5000.

[0017] It also includes the preparation of reference solutions, including Tianlong reference medicinal material, pterin-6-carboxylic acid reference standard, hypoxanthine reference standard, and thymine reference standard;

[0018] The preparation method of the Tianlong reference medicinal material reference solution is as follows: take Tianlong reference medicinal material, add water, sonicate, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution;

[0019] The preparation method of the reference solutions of pterin-6-carboxylic acid, hypoxanthine, and thymine is as follows: take pterin-6-carboxylic acid, hypoxanthine, and thymine reference standards, and add water or ammonia solution to prepare reference solutions.

[0020] The feature spectrum contains 8 feature peaks with relative retention times as follows:

[0021] Peak 1: 0.38, Peak 2: 0.42, Peak 3: 0.52, Peak 4: 0.65, Peak 5: 1.0, Peak 6: 1.06, Peak 7: 1.25, Peak 8: 1.45, with relative retention times fluctuating within ±10%.

[0022] Peak 3 is hypoxanthine; peak 4 is thymine; peak 5 is pterin-6-carboxylic acid.

[0023] The Tianlong or its preparations include Tianlong medicinal materials, processed slices, standard decoctions, and formulated granules.

[0024] This invention also provides a method for identifying adulterants of Tianlong medicinal materials, which includes the following steps:

[0025] a. Weigh the sample to be tested;

[0026] b. Detect the characteristic peaks according to the method for constructing the characteristic spectrum of Tianlong or its preparations. If the 8 characteristic peaks are completely consistent, it is genuine Tianlong medicinal material.

[0027] The aforementioned adulterated medicinal material is gecko.

[0028] The method for constructing characteristic spectra in this invention uses *Tianlong* (a type of medicinal herb) and pterin-6-carboxylic acid as reference materials to determine eight characteristic peaks. Peak 3 is identified as hypoxanthine, peak 4 as thymine, and peak 5 as pterin-6-carboxylic acid. These eight characteristic peaks are stably present in *Tianlong* medicinal materials, processed slices, standard decoctions, and formulated granules, indicating that the material basis of *Tianlong* medicinal materials, processed slices, standard decoctions, and formulated granules is consistent and all originates from *Tianlong* medicinal materials. This method can effectively and comprehensively reflect the condition of *Tianlong* and its preparations, and can distinguish them from other easily confused substances (such as gecko). Attached Figure Description

[0029] Figure 1 Extraction solvent investigation;

[0030] Figure 2 Examining the extraction method;

[0031] Figure 3 Extraction time consideration;

[0032] Figure 4 Investigation of solvent addition amount;

[0033] Figure 5 Specific overlay image;

[0034] Figure 6 Spectrum of pterin-6-carboxylic acid reference standard;

[0035] Figure 7 Spectrum of pterin-6-carboxylic acid sample;

[0036] Figure 8 Spectrum of thymine reference standard;

[0037] Figure 9 Spectral diagram of thymine sample;

[0038] Figure 10 Spectral overlay of hypoxanthine reference standard;

[0039] Figure 11 Spectral overlay of hypoxanthine samples;

[0040] Figure 12 Comparison of characteristic spectra of Tianlong medicinal materials using different instruments;

[0041] Figure 13 Different chromatographic columns;

[0042] Figure 14Characteristic atlases of 16 batches of Tianlong medicinal materials (Note: Each atlas represents a batch number as follows: S1: XXLS202307797; S2: XXLS202307798; S3: XXLS202307799; S4: XXLS202307800; S5: XXLS202307801; S6: XXLS202307802; S7: XXLS202307803; S8: XXLS202...). 307804; S9: XXLS202307805; S10: XXLS202307806; S11: XXLS202307807; S12: XXLS20230780 8; S13: XXLS202307809; S14: XXLS202307810; S15: XXLS202307811; S16: 010444-2307001);

[0043] Figure 15 Characteristic chromatogram of Tianlong medicinal material (peak 3: hypoxanthine; peak 4: thymine; peak 5 (S): pterin-6-carboxylic acid; chromatographic column: Shim-pack Scepter C18-120 4.6×250mm, 5μm);

[0044] Figure 16 Characteristic chromatograms of 16 batches of Tianlong medicinal slices (Note: Each chromatogram represents a batch number as follows: S1: TL-230701; S2: TL-230702; S3: TL-230703; S4: TL-230704; S5: TL-230705; S6: TL-230706; S7: TL-230707; S8: TL-230708; S9: TL-230709; S10: TL-230710; S11: TL-230711; S12: TL-230712; S13: TL-230713; S14: TL-230714; S15: TL-230715; S16: TL-230716).

[0045] Figure 17 Characteristic chromatogram of Tianlong medicinal slices (peak 3: hypoxanthine; peak 4: thymine; peak 5 (S): pterin-6-carboxylic acid; chromatographic column: Shim-pack Scepter C18-120 4.6×250mm, 5μm);

[0046] Figure 18 Chromatographic peak identification;

[0047] Figure 19 Spectrum of pterin-6-carboxylic acid reference standard;

[0048] Figure 20 Spectrum of pterin-6-carboxylic acid sample;

[0049] Figure 21 Spectrum of thymine reference standard;

[0050] Figure 22 Spectral diagram of thymine sample;

[0051] Figure 23 Spectral overlay of hypoxanthine reference standard;

[0052] Figure 24 Spectral overlay of hypoxanthine samples;

[0053] Figure 25 Comparison of characteristic spectra of Tianlong standard decoction using different instruments;

[0054] Figure 26 Different chromatographic columns;

[0055] Figure 27 Overlay diagram of Tianlong standard decoction characteristics (batch numbers from bottom to top are: TL-BT-230701, TL-BT-230702, TL-BT-230703, TL-BT-230704, TL-BT-230705, TL-BT-230706, TL-BT-230707, TL-BT-230708, TL-BT-230709, TL-BT-230710, TL-BT-230711, WFG-BT-210912, TL-BT-230713, TL-BT-230714, TL-BT-230715, TL-BT-230716);

[0056] Figure 28 Characteristic chromatogram of Tianlong standard decoction (peak 3: hypoxanthine; peak 4: thymine; peak 5 (S): pterin-6-carboxylic acid; chromatographic column: Shim-pack Scepter C18-120 4.6×250mm, 5μm);

[0057] Figure 29 UV absorption spectrum of pterin-6-carboxylic acid;

[0058] Figure 30 Thymine ultraviolet absorption spectrum;

[0059] Figure 31 Ultraviolet absorption spectrum of hypoxanthine;

[0060] Figure 32 Chromatograms of Tianlong formula granules at different wavelengths;

[0061] Figure 33 Chromatograms were obtained by examining the column temperature.

[0062] Figure 34 Flow rate analysis of chromatograms;

[0063] Figure 35 Extraction solvent investigation;

[0064] Figure 36 Examining the extraction method;

[0065] Figure 37 Extraction time consideration;

[0066] Figure 38 Investigation of solvent addition amount;

[0067] Figure 39 Specific overlay image;

[0068] Figure 40 Spectrum of pterin-6-carboxylic acid reference standard;

[0069] Figure 41 Spectrum of pterin-6-carboxylic acid sample;

[0070] Figure 42 Spectrum of thymine reference standard;

[0071] Figure 43 Spectral diagram of thymine sample;

[0072] Figure 44 Spectral overlay of hypoxanthine reference standard;

[0073] Figure 45 Spectral overlay of hypoxanthine samples;

[0074] Figure 46 Comparison of characteristic spectra of different Tianlong finished products;

[0075] Figure 47 Different chromatographic columns;

[0076] Figure 48 Characteristic spectrum verification diagrams of three batches of Tianlong formula granules;

[0077] Figure 49 Tianlong Formula Granules Comparison Characteristic Chromatography (Peak 3: Hypoxanthine; Peak 4: Thymine; Peak 5 (S): Pterin-6-carboxylic acid; Column: Shim-pack Scepter C18-120 4.6×250mm, 5μm);

[0078] Figure 50 Chromatogram using methanol-0.15% phosphoric acid solution as the mobile phase;

[0079] Figure 51 Comparison chart of Tianlong and Gecko granules. Detailed Implementation

[0080] Example 1: Detection of the Characteristic Spectrum of Tianlong Medicinal Herbs according to the Present Invention

[0081] The 1994 edition of the "Shanghai Municipal Standards for Traditional Chinese Medicine" defines "Tianlong" as the dried whole body of the multi-warted gecko *Gekkojaponicas* (Dumeril et Bibron), a species of gecko. It is captured in summer and autumn, lured at night using lights, killed, and then dried over a low flame or in the sun, or its internal organs are removed, and it is then dried using thin bamboo strips.

[0082] To ensure the reliability of the established Tianlong quality standards, a total of 16 batches of Tianlong medicinal materials were collected from Tongling City, Anhui Province, Zhengzhou City, Henan Province, and Thailand. Specific origin information is shown in Table 1.

[0083] Table 1 Summary Table of Tianlong Medicinal Herb Origin Information

[0084]

[0085] 1. Instruments and Materials

[0086] High-performance liquid chromatographs: Shimadzu LC-20AD high-performance liquid chromatograph, Waters e2695 high-performance liquid chromatograph;

[0087] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0088] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0089] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0090] Acetonitrile and acetic acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0091] Pterin-6-carboxylic acid (Shanghai Shidander Standard Technical Service Co., Ltd., batch number: 15847, content calculated as 99.8%);

[0092] Xanthine (China National Institutes for Food and Drug Control, batch number: 140661-202005, content calculated as 99.4%);

[0093] Thymine (China National Institutes for Food and Drug Control, batch number: 140708-201902, content calculated as 99.7%);

[0094] Tianlong reference medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 390016-202306);

[0095] Tianlong Medicinal Herbs (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: XXLS202307797, XXLS202307798, XXLS202307799, XXLS202307801, XXLS202307802, XXLS202307803, XXLS202307804, XXLS202307805, XXLS202307806, XXLS202307807, XXLS202307808, XXLS202307809, XXLS202307810, XXLS202307811, 010444-2307001).

[0096] 2 Feature Map Detection Method

[0097] The chromatographic conditions for the characteristic chromatograms of Tianlong medicinal materials are referenced in document 7, which details the method for chromatographic analysis of Tianlong formula granules, as follows:

[0098] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 289 nm. The theoretical plate number, calculated based on the pterin-6-carboxylic acid peak, should be no less than 5000.

[0099]

[0100] 3. Preparation and Investigation of Test Samples

[0101] 3.1 Investigation of extraction solvent

[0102] Accurately weigh approximately 1.0g of *Tianlong* medicinal herb powder (passed through a No. 3 sieve) (batch number: XXLS202307808), place it in a stoppered conical flask, and extract with water, 0.028% ammonia, 10% methanol, 70% methanol, methanol, and 10% ethanol respectively. Accurately add 25ml of water to each extract, seal tightly, weigh, and sonicate (600W, 40kHz) for 30 minutes. Cool, weigh again, and replenish the lost weight with the extraction solvent. Shake well, filter, and collect the filtrate. See [link to flask description]. Figure 1 .

[0103] The results showed that water was used as the extraction solvent, resulting in a larger amount of chromatographic peak information and a better peak shape. Therefore, water was determined to be the extraction solvent for the test sample.

[0104] 3.2 Investigation of the preparation and extraction methods of the test sample

[0105] Take approximately 1.0 g of Tianlong medicinal material powder (passed through a No. 3 sieve) (batch number: XXLS202307808), place it in a stoppered conical flask, add 25 ml of water, seal tightly, and investigate the extraction methods for the sample: reflux and ultrasonic (600 W power, 40 kHz frequency). Extraction time is 30 minutes. After cooling, shake well, filter, and collect the filtrate. For water extraction, add 25 ml of water, boil for half an hour, filter, evaporate to dryness, add 25 ml of water to the residue, ultrasonically treat (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate. Results are shown below. Figure 2 .

[0106] The results showed that the chromatographic effects of ultrasonic extraction and reflux extraction were basically the same, and ultrasonic extraction was superior to ultrasonic extraction after boiling in water. Therefore, ultrasonic extraction was selected as the extraction method for the test sample in this experiment.

[0107] 3.3 Examination of extraction time

[0108] Take approximately 1.0 g of Tianlong medicinal herb powder (passed through a No. 3 sieve) (batch number: XXLS202307808), place it in a stoppered conical flask, add 25 ml of water, seal tightly, and test the sample by ultrasonic treatment (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to product]. Figure 3 .

[0109] The results showed that the chromatograms were basically consistent under different extraction time conditions. To ensure sufficient extraction, the extraction time for the test sample was determined to be 30 minutes.

[0110] 3.4 Investigation of Solvent Addition Amount

[0111] Take approximately 1.0g of Tianlong medicinal herb powder (passed through a No. 3 sieve) (batch number: XXLS202307808), place it in a stoppered conical flask, add 10ml, 25ml, and 50ml of water respectively, seal tightly, and sonicate (600W power, 40kHz frequency) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the final product. See [link to product description]. Figure 4 .

[0112] The results showed that the peak areas of the characteristic chromatograms were moderate when the solvent volume was 25 ml. Therefore, the solvent volume for the test sample was determined to be 25 ml.

[0113] 3.5 Determine the preparation method of the test sample

[0114] Take an appropriate amount of Tianlong medicinal powder (passed through a No. 3 sieve), about 1.0g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of water, seal tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, add water to make up the lost weight, shake well, filter it, and collect the filtrate to obtain the product.

[0115] 4. Methodological Investigation

[0116] 4.1 Chromatographic Peak Identification

[0117] Preparation of the test solution: Prepare the Tianlong medicinal material test solution according to the experimental conditions proposed above.

[0118] Preparation of reference solution: Take appropriate amounts of pterin-6-carboxylic acid reference standard, hypoxanthine reference standard, and thymine reference standard, accurately weigh them, and add water to prepare a solution containing 40 μg per ml.

[0119] Preparation of Tianlong reference medicinal material solution: Take about 1.0g of Tianlong reference medicinal material, place it in a stoppered conical flask, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution.

[0120] Preparation of negative control solution: Prepare negative control solution of Tianlong medicinal material according to the experimental conditions proposed above.

[0121] The characteristic spectral peaks of Tianlong medicinal materials were located. (See...) Figure 5-11 .

[0122] The results showed that the retention times and spectra of pterin-6-carboxylic acid, thymine, and hypoxanthine reference standards were consistent with those of the target peaks in Tianlong medicinal materials. Figure 1 The method exhibits good specificity, with a one-to-one correspondence and no interference from the negative solution.

[0123] In summary, eight characteristic peaks from medicinal materials were included in subsequent investigations, and methodological studies were conducted.

[0124] 4.2 Repeatability Test

[0125] Six portions of Tianlong medicinal material (batch number: XXLS202307808) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 2.

[0126] Table 2 Repeatability Tests - Relative Retention Time Ratios

[0127]

[0128] 4.3 Intermediate Precision Examination

[0129] Based on the above-specified experimental conditions, 12 portions of Tianlong medicinal material were accurately weighed to prepare test solutions, which were then analyzed using a Shimadzu LC-20AD and a Waters e2695 high-performance liquid chromatograph, respectively. (See...) Figure 12 Table 3.

[0130] Table 3 Intermediate Precision-Relative Retention Time Ratio

[0131]

[0132] 4.4 Durability Test

[0133] Based on the above-planned experimental conditions, the chromatographic column was tested as follows: Shim-pack Scepter C18-120 4.6×250mm, 5μm (column 1). HSS T3 4.6×250mm, 5μm (column 2) The study was conducted using an AQ-C18 column (4.6 × 250 mm, 5 μm) and the results are shown in [reference needed]. Figure 13 Table 4.

[0134] Table 4. Column robustness study - relative retention time

[0135]

[0136] The results showed that the relative retention time (RSD) values ​​varied significantly when using different chromatographic columns. The Shim-pack Scepter C18-120 (4.6 × 250 mm, 5 μm) is recommended.

[0137] 4.5 Stability Test

[0138] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h, respectively. See Table 5.

[0139] Table 5 Stability Study - Retention Time

[0140]

[0141] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.04% and 0.12%, and the sample solution was relatively stable within 24 hours.

[0142] In summary, the retention times and relative retention times of each characteristic peak meet the requirements in all the above tests, indicating that this method is effective.

[0143] 5. Methodological Summary

[0144] When peak 5 is selected as the S peak, the RSD of the retention time or relative retention time of each stage is shown in Table 6.

[0145] Table 6 Summary of RSD values ​​when peak 5 is an S-peak

[0146]

[0147] The results show that, except for robustness, the retention times or RSD values ​​of the relative retention times of each characteristic peak meet the requirements in all the above tests, indicating that the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0148] 6. Determination of characteristic peaks and establishment of reference spectra

[0149] Using this method, characteristic spectral analysis was performed on 19 batches of samples, and the relative retention time and relative peak area ratio were calculated. See [link / reference]. Figure 14 Table 7.

[0150] Table 7. Relative retention times of 16 batches of Tianlong medicinal materials

[0151]

[0152]

[0153] Based on the principles of stable relative retention times, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. The specifications are as follows: the chromatogram of the test sample should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peak 5 should correspond to the retention time of the reference standard peak. The peak corresponding to the pterin-6-carboxylic acid reference standard peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.38 (peak 1), 0.42 (peak 2), 0.52 (peak 3), 0.65 (peak 4), 1.06 (peak 6), 1.25 (peak 7), and 1.45 (peak 8).

[0154] The above samples were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and a reference characteristic chromatogram of Tianlong medicinal material was established. (See attached image.) Figure 15 .

[0155] Example 2: Detection of the Characteristic Spectrum of Tianlong Herbal Pieces according to the present invention

[0156] 1. Experimental Instruments and Materials

[0157] High-performance liquid chromatograph: Shimadzu LC-20AD high-performance liquid chromatograph;

[0158] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0159] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0160] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0161] Acetonitrile and acetic acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0162] Pterin-6-carboxylic acid (Shanghai Shidander Standard Technical Service Co., Ltd., batch number: 15847, content calculated as 99.8%)

[0163] Hypoxanthine (China National Institutes for Food and Drug Control, batch number: 140661-202005, content calculated as 99.4%)

[0164] Thymine (China National Institutes for Food and Drug Control, batch number: 140708-201902, content calculated as 99.7%)

[0165] Tianlong reference medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 390016-202306),

[0166] Tianlong Herbal Slices (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: TL-230701, TL-230702, TL-230703, TL-230704, TL-230705, TL-230706, TL-230707, TL-230708, TL-230709, TL-230710, TL-230711, TL-230712, TL-230713, TL-230714, TL-230715, TL-230716).

[0167] 2 Chromatographic conditions

[0168] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 ml / min; column temperature 30℃; detection wavelength 260 nm.

[0169] The theoretical plate number, calculated based on the pterin-6-carboxylic acid peak, should be no less than 5000.

[0170]

[0171] Preparation of the reference solution: Take 1.0 g of *Tianlong* reference material, place it in a stoppered conical flask, add 25 ml of water, sonicate (600 W, 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Accurately weigh an appropriate amount of pterin-6-carboxylic acid reference standard, add aqueous solution to prepare a solution containing 40 μg per ml.

[0172] Preparation of the test solution: Take an appropriate amount of the powder (passed through a No. 3 sieve), about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of water, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake well, filter it, and take the filtrate to obtain the test solution.

[0173] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0174] 2.2.2.3 Verification results of 16 batches of Tianlong medicinal slices

[0175] Using this method, characteristic spectral analysis was performed on 16 batches of samples, and the relative retention time and relative peak area ratio were calculated. See Figure 16 Table 8.

[0176] Table 8. Relative retention time of 16 batches of Tianlong medicinal slices

[0177]

[0178] Based on the principles of stable relative retention times, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. The specifications are as follows: the chromatogram of the test sample should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peak 5 should correspond to the retention time of the reference standard peak. The peak corresponding to the pterin-6-carboxylic acid reference standard peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.38 (peak 1), 0.42 (peak 2), 0.52 (peak 3), 0.65 (peak 4), 1.06 (peak 6), 1.25 (peak 7), and 1.45 (peak 8).

[0179] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 19 batches of Tianlong (Hemiberlesia lingua) slices, and a reference chromatogram of the characteristic chromatograms of Tianlong slices was established. See [link / reference]. Figure 17 .

[0180] Example 3: Detection of the characteristic chromatogram of the Tianlong standard decoction of the present invention.

[0181] 1. Experimental Instruments and Materials

[0182] High-performance liquid chromatographs: Shimadzu LC-20AD high-performance liquid chromatograph, Waters e2695 high-performance liquid chromatograph;

[0183] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0184] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0185] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0186] Column: Shim-pack Scepter C18-120 4.6×250mm, 5μm HSS T3 4.6×250mm, 5μm AQ-C18 4.6×250mm, 5μm.

[0187] Acetic acid and acetonitrile were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0188] Pterin-6-carboxylic acid (Shanghai Shidander Standard Technical Service Co., Ltd., batch number: 15847, content calculated as 99.8%);

[0189] Hypoxanthine (China National Institutes for Food and Drug Control, batch number: 140661-202005, content calculated as 99.4%);

[0190] Thymine (China National Institutes for Food and Drug Control, batch number: 140708-201902, content calculated as 99.7%);

[0191] Tianlong reference medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 390016-202306);

[0192] Tianlong Standard Decoction Freeze-Dried Powder (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: TL-BT-230701, TL-BT-230702, TL-BT-230703, TL-BT-230704, TL-BT-230705, TL-BT-230706, TL-BT-230707, TL-BT-230708, TL-BT-230709, TL-BT-230710, TL-BT-230711, TL-BT-230712, TL-BT-230713, TL-BT-230714, TL-BT-230715, TL-BT-230716).

[0193] 2 Feature Detection Methods

[0194] The chromatographic conditions and preparation methods for the characteristic chromatogram of Tianlong standard decoction are detailed in reference 7, specifically the method for the characteristic chromatogram of Tianlong formula granules:

[0195] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 ml / min; column temperature 30℃; detection wavelength 260 nm.

[0196] The theoretical plate number, calculated based on the pterin-6-carboxylic acid peak, should be no less than 5000.

[0197]

[0198] Preparation of the reference solution: Take 1.0 g of *Tianlong* reference material, place it in a stoppered conical flask, add 25 ml of water, sonicate (600 W, 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Accurately weigh an appropriate amount of pterin-6-carboxylic acid reference standard, add 0.028% ammonia solution to prepare a solution containing 20 μg per ml.

[0199] Preparation of the test solution: Weigh approximately 0.5 g of this product accurately, place it in a stoppered conical flask, add 25 ml of water accurately, stopper tightly, weigh, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, replenish the lost weight with water, shake well, filter, and collect the filtrate to obtain the test solution.

[0200] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0201] 3. Methodological Investigation

[0202] 3.1 Chromatographic Peak Identification

[0203] Preparation of the test solution: Prepare the Tianlong standard decoction test solution according to the experimental conditions proposed above.

[0204] Preparation of reference solution: Accurately weigh pterin-6-carboxylic acid reference standard, add 0.028% ammonia solution to prepare a solution containing 20 μg per ml. Separately, accurately weigh appropriate amounts of hypoxanthine reference standard and thymine reference standard, add water to prepare a solution containing 40 μg per ml.

[0205] Preparation of Tianlong reference medicinal material solution: Take about 1.0g of Tianlong reference medicinal material, place it in a stoppered conical flask, add 25ml of water, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material.

[0206] Preparation of negative control solution: Prepare negative control solution of Tianlong standard decoction according to the experimental conditions proposed above.

[0207] The characteristic peaks of Tianlong standard decoction were located. (See...) Figure 18-24 .

[0208] The results showed that the retention times and spectra of pterin-6-carboxylic acid, thymine, and hypoxanthine reference standards were consistent with those of the target peaks in Tianlong standard decoction. Figure 1 The method exhibits good specificity, with a one-to-one correspondence and no interference from the negative solution.

[0209] 3.2 Repeatability Test

[0210] Accurately weigh 6 portions of Tianlong standard decoction (batch number: TL-BT-230704), and prepare and determine the solution according to the proposed experimental method. See Table 9.

[0211] Table 9 Repeatability Tests - Relative Retention Time

[0212]

[0213] The results show that the relative retention times of each characteristic peak are consistent, with a relative retention time RSD of 0.00%–1.30%. The method exhibits good repeatability.

[0214] 3.3 Intermediate Precision Examination

[0215] Based on the above-planned experimental conditions, 12 portions of Tianlong standard decoction (batch number: TL-BT-230704) were accurately weighed to prepare test solutions, which were then analyzed using a Shimadzu LC-20AD and a Waters e2695 high-performance liquid chromatograph, respectively. See Figure 25 Table 10.

[0216] Table 10 Intermediate Precision-Relative Retention Time Ratio

[0217]

[0218] 3.4 Durability Test

[0219] Based on the above-planned experimental conditions, the chromatographic column was tested as follows: Shim-pack Scepter C18-120 4.6×250mm, 5μm (column 1). HSS T3 4.6×250mm, 5μm (column 2) The study was conducted using an AQ-C18 column (4.6 × 250 mm, 5 μm) and the results are shown in [reference needed]. Figure 26 Table 11.

[0220] Table 11 Column robustness study - relative retention time

[0221]

[0222] The results showed that the relative retention time (RSD) values ​​varied significantly when using different chromatographic columns. The Shim-pack Scepter C18-120 (4.6 × 250 mm, 5 μm) is recommended.

[0223] 3.5 Stability Test

[0224] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h, respectively. See Table 12.

[0225] Table 12 Stability Study - Retention Time

[0226]

[0227]

[0228] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.04% and 0.41%, and the sample solution was relatively stable within 24 hours.

[0229] 4. Methodological Summary

[0230] When peak 5 is selected as the S peak, the RSD of the retention time or relative retention time of each item is shown in Table 13.

[0231] Table 13 Summary of RSD values ​​for retention time or relative retention time

[0232]

[0233] The results show that the retention times or RSD values ​​of the relative retention times of each characteristic peak meet the requirements in all the above investigations, indicating that the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0234] 5. Determination of characteristic peaks and establishment of reference spectra

[0235] Using this method, characteristic spectral analysis was performed on 16 batches of samples, and the relative retention time and relative peak area ratio were calculated. See Figure 27 Table 14.

[0236] Table 14 Relative Retention Times of 16 Batches of Tianlong Standard Decoction

[0237]

[0238] Based on the principles of stable relative retention times, detectability in all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. The specifications are as follows: the chromatogram of the test sample should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peak 5 should correspond to the retention time of the reference standard peak. The peak corresponding to the pterin-6-carboxylic acid reference standard peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.38 (peak 1), 0.42 (peak 2), 0.52 (peak 3), 0.65 (peak 4), 1.06 (peak 6), 1.25 (peak 7), and 1.45 (peak 8).

[0239] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 16 batches of Tianlong standard decoction, and a reference chromatogram of the characteristic chromatograms of Tianlong standard decoction was established. See [link / reference]. Figure 28 .

[0240] Example 4: Detection of the Characteristic Spectrum of the Tianlong Formula Granules of the Present Invention

[0241] 1. Experimental Instruments and Materials

[0242] High-performance liquid chromatographs: Shimadzu LC-20AD high-performance liquid chromatograph, Waters e2695 high-performance liquid chromatograph;

[0243] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0244] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0245] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0246] Column: Shim-pack Scepter C18-120 4.6×250mm, 5μm HSS T3 4.6×250mm, 5μm AQ-C18 4.6×250mm, 5μm.

[0247] Acetic acid and acetonitrile were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0248] Pterin-6-carboxylic acid (Shanghai Shidander Standard Technical Service Co., Ltd., batch number: 15847, content calculated as 99.8%);

[0249] Hypoxanthine (China National Institutes for Food and Drug Control, batch number: 140661-202005, content calculated as 99.4%);

[0250] Thymine (China National Institutes for Food and Drug Control, batch number: 140708-201902, content calculated as 99.7%);

[0251] Tianlong reference medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 390016-202306);

[0252] Tianlong Formula Granules (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: 18110027, 2308101, 2308102, 2308103).

[0253] 2 Chromatographic conditions

[0254] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate of 1.0 mL / min; column temperature of 30 °C; and detection wavelength of 260 nm. The theoretical plate number, calculated based on the pterin-6-carboxylic acid peak, should be no less than 5000.

[0255]

[0256] 2.1 Wavelength Selection

[0257] Based on the above-planned experimental conditions, a diode array detector was used to perform a full-band scan of the Tianlong test solution, and chromatograms of the test solution at wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, and 300 nm were extracted, as shown in the figures. Figure 29-32 .

[0258] The results showed that the chromatographic peak information was large and the chromatographic baseline was stable at a detection wavelength of 260 nm, so the detection wavelength was determined to be 260 nm.

[0259] 2.2 Column Temperature Investigation

[0260] Based on the above-specified experimental conditions, the results were investigated at column temperatures of 25℃, 30℃, and 35℃. (See...) Figure 33 Table 15.

[0261] Table 15 Column Temperature Study - Relative Retention Time

[0262]

[0263] The results showed that the chromatogram peaks were more symmetrical and the retention time was more suitable at 30℃, so the column temperature was selected as 30℃.

[0264] 2.3 Flow velocity investigation

[0265] Based on the above-established experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. (See...) Figure 34 Table 16.

[0266] Table 16 Flow velocity study - relative retention time

[0267]

[0268] The results showed that the chromatogram peaks were relatively symmetrical and the retention time was appropriate at a flow rate of 1.0 ml / min, so a flow rate of 1.0 ml / min was selected.

[0269] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of Tianlong formula granules were determined as follows: Octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 ml / min; column temperature 30℃; detection wavelength 260 nm. The theoretical plate number, calculated based on the pterin-6-carboxylic acid peak, should not be less than 5000.

[0270]

[0271] 3. Preparation and Investigation of Test Samples

[0272] 3.1 Investigation of extraction solvent

[0273] Take approximately 0.5g of Tianlong formula granules (batch number: 18110027), grind them into a fine powder, and accurately weigh the powder. Place it in a stoppered conical flask and extract with water, 0.028% ammonia, 10% methanol, 70% methanol, methanol, and 10% ethanol, respectively. Accurately add 25ml of each extract to the flask, seal tightly, weigh, and sonicate (600W, 40kHz) for 30 minutes. After cooling, weigh again, replenish the lost weight with the extraction solvent, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 35 .

[0274] The results showed that water was used as the extraction solvent, resulting in a larger amount of chromatographic peak information and a better peak shape. Therefore, water was determined to be the extraction solvent for the test sample.

[0275] 3.2 Examination of Extraction Methods

[0276] Take an appropriate amount of Tianlong formula granules (batch number: 18110027), grind them into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of water, seal tightly, and test the extraction methods for the sample: reflux and ultrasonication (power 600W, frequency 40kHz). The extraction time is 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the sample. See Figure 36 .

[0277] The results showed that the chromatographic effects were basically the same when using ultrasonic extraction and reflux extraction. Ultrasonic extraction was chosen as the extraction method for the test sample in this experiment.

[0278] 3.3 Examination of extraction time

[0279] Take an appropriate amount of Tianlong formula granules (batch number: 18110027), grind them into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of water, seal tightly, and test the sample by ultrasonic treatment (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to product]. Figure 37 .

[0280] The results showed that the chromatograms were basically consistent under different extraction time conditions. To ensure sufficient extraction, the extraction time for the test sample was determined to be 30 minutes.

[0281] 3.4 Investigation of Solvent Addition Amount

[0282] Take an appropriate amount of Tianlong formula granules (batch number: 18110027), grind them into a fine powder, and take about 0.5g. Place them in stoppered conical flasks, add 10ml, 25ml, and 50ml of water respectively, seal tightly, and sonicate (power 600W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, and collect the filtrate. See [link to product]. Figure 38 .

[0283] The results showed that the peak areas of the characteristic chromatograms were moderate when the solvent volume was 25 ml. Therefore, the solvent volume for the test sample was determined to be 25 ml.

[0284] 3.5 Determine the preparation method of the test sample

[0285] Take an appropriate amount of Tianlong formula granules, grind them into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of water, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0286] 3.6 Feature Mapping Method

[0287] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0288] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 ml / min; column temperature 30℃; detection wavelength 260 nm.

[0289] The theoretical plate number, calculated based on the pterin-6-carboxylic acid peak, should be no less than 5000.

[0290]

[0291] Preparation of the reference solution: Take 1.0 g of *Tianlong* reference material, place it in a stoppered conical flask, add 25 ml of water, sonicate (600 W, 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Accurately weigh an appropriate amount of pterin-6-carboxylic acid reference standard, add 0.028% ammonia solution to prepare a solution containing 20 μg per ml.

[0292] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of water, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with water, shake well, filter it, and take the filtrate to obtain the test solution.

[0293] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0294] 4. Methodological Investigation

[0295] 4.1 Chromatographic Peak Identification

[0296] Preparation of the test solution: Prepare the Tianlong formula granule test solution according to the experimental conditions proposed above.

[0297] Preparation of reference solution: Accurately weigh pterin-6-carboxylic acid reference standard, add 0.028% ammonia solution to prepare a solution containing 20 μg per ml. Separately, accurately weigh appropriate amounts of hypoxanthine reference standard and thymine reference standard, add water to prepare a solution containing 40 μg per ml.

[0298] Preparation of Tianlong reference medicinal material solution: Take about 1.0g of Tianlong reference medicinal material, place it in a stoppered conical flask, add 25ml of water, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material.

[0299] Preparation of negative control solution: Prepare negative control solution for Tianlong formula granules according to the experimental conditions proposed above.

[0300] The characteristic peaks of the Tianlong formula granules were located. See [link / reference]. Figures 39-45 .

[0301] The results showed that the retention times and spectra of pterin-6-carboxylic acid, thymine, and hypoxanthine reference standards were consistent with the retention times and spectra of the target peaks in Tianlong formula granules. Figure 1 The method exhibits good specificity, with a one-to-one correspondence and no interference from the negative solution.

[0302] 4.2 Repeatability Test

[0303] Six portions of Tianlong formula granules were accurately weighed and prepared and measured according to the proposed experimental method. See Table 17.

[0304] Table 17 Repeatability Tests - Relative Retention Time Ratios

[0305]

[0306] The results show that the relative retention time RSD values ​​of each characteristic peak are 0.00%-1.37%, indicating that the method has good repeatability.

[0307] 4.3 Intermediate Precision Examination

[0308] Based on the above-specified experimental conditions, 12 portions of Tianlong formula granules were accurately weighed to prepare test solutions, which were then analyzed using a Shimadzu LC-20AD and a Waters e2695 high-performance liquid chromatograph, respectively. (See...) Figure 46 Table 18.

[0309] Table 18 Intermediate Precision - Relative Retention Time

[0310]

[0311] 4.4 Durability Test

[0312] Based on the above-planned experimental conditions, the chromatographic column was tested as follows: Shim-pack Scepter C18-120 4.6×250mm, 5μm (column 1). HSS T3 4.6×250mm, 5μm (column 2) The study was conducted using an AQ-C18 column (4.6 × 250 mm, 5 μm) and the results are shown in [reference needed]. Figure 47 Table 19.

[0313] Table 19 Column Robustness Study - Relative Retention Time

[0314]

[0315] The results showed that the relative retention time (RSD) values ​​varied significantly when using different chromatographic columns. The Shim-pack Scepter C18-120 (4.6 × 250 mm, 5 μm) is recommended.

[0316] 4.5 Stability

[0317] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h. See Table 20.

[0318] Table 20 Stability Study - Retention Time

[0319]

[0320] The results showed that the RSD values ​​of the corresponding characteristic peak retention times were 0.03% to 0.12%, and the sample solution was relatively stable within 24 hours.

[0321] 5. Methodological Summary

[0322] When peak 5 is selected as the S peak, the RSD of the retention time or relative retention time of each item is shown in Table 21.

[0323] Table 21 Summary of RSD values ​​for retention time or relative retention time

[0324]

[0325]

[0326] The results showed that the RSD values ​​of the relative retention times of each characteristic peak were relatively large under different column temperatures, flow rates, and column conditions. Therefore, flow rates and column temperatures are temporarily specified, and the recommended column is suggested for testing. All other conditions were met, indicating the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0327] 6. Determination of characteristic peaks and establishment of reference spectra

[0328] 6.1 Validation results of three batches of Tianlong formula granules

[0329] The characteristic spectra of three batches of this product were determined using the proposed method, and the relative retention times and relative peak areas were calculated. (See attached image.) Figure 48 Table 22.

[0330] Table 22 Relative Retention Times of Three Batches of Tianlong Formula Granules

[0331]

[0332] Based on the principles of stable relative retention times, detectability across all batches of samples, and relatively high peak values, eight peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 5 was designated as the S peak, the relative retention times (RSDs) of all eight characteristic peaks in the three batches of Tianlong formula granules were less than 2%.

[0333] 6.2 Establishment of Limits for Relative Retention Time

[0334] Consistent with the Tianlong standard decoction, the final specification is as follows: the chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 5 should correspond to the retention time of the reference peak. The peak corresponding to the peak of the pterin-6-carboxylic acid reference standard is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.38 (peak 1), 0.42 (peak 2), 0.52 (peak 3), 0.65 (peak 4), 1.06 (peak 6), 1.25 (peak 7), and 1.45 (peak 8).

[0335] Three batches of Tianlong formula granules were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version). A reference chromatogram of the characteristic chromatogram of Tianlong formula granules was established. (See attached image) Figure 49 .

[0336] Comparative Example 1: Experimental results using methanol-0.15% phosphoric acid solution as the mobile phase (the mobile phase in the prior art).

[0337] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.15% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 260 nm. The theoretical plate number, calculated based on the pterin-6-carboxylic acid peak, should be no less than 5000.

[0338]

[0339] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of water, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with water, shake well, filter it, and take the filtrate to obtain the test solution.

[0340] The assay involves precisely pipetting 10 μl of the test solution and injecting it into the liquid chromatograph for determination.

[0341] See results Figure 50 .

[0342] The results showed that when methanol-0.15% phosphoric acid solution was used as the mobile phase, the chromatographic peak resolution was poor and the overall appearance was not aesthetically pleasing, making it difficult to use as a characteristic chromatographic method for asparagus preparations.

[0343] Comparative Example 2: Study on Easily Confused Products

[0344] Market research indicates that gecko is a common adulterant of Tianlong medicinal materials. After water extraction, Tianlong preparations lose their original appearance, making them even more difficult to distinguish from genuine products. This characteristic chromatographic method was used to detect Tianlong and gecko granules in formulations. The specific method is as follows:

[0345] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 260 nm.

[0346]

[0347] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of water, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with water, shake well, filter it, and take the filtrate to obtain the test solution.

[0348] The assay involves precisely pipetting 10 μl of the test solution and injecting it into the liquid chromatograph for determination.

[0349] See results Figure 51 .

[0350] The results showed that under the chromatographic conditions of this method, the chromatograms of Tianlong granules and gecko differed significantly; almost none of the eight characteristic peaks of Tianlong were present in the chromatogram of gecko. Therefore, this method makes it relatively easy to distinguish between Tianlong and gecko products.

[0351] This invention presents a novel characteristic spectral method. The sample preparation method is simple and easy to operate, and it identifies numerous characteristic peaks, enabling accurate and reliable detection of *Tianlong* (a type of medicinal herb) and its preparations. It effectively controls the authenticity (authenticity means it originates from *Tianlong* and is not derived from other easily confused medicinal materials), quality consistency (consistency means that *Tianlong* raw materials, processed slices, standard decoctions, and formulated granules all exhibit eight characteristic peaks, indicating a fundamentally consistent material basis, all originating from *Tianlong* raw materials), and stability of *Tianlong* and its preparations. This provides a basis for comprehensively evaluating the quality of *Tianlong* and its preparations.

Claims

1. A method for constructing a characteristic map of a Tianlong or a preparation thereof, characterized by: It comprises the following steps: a. Preparation of test sample solution: Take the test sample of Tianlong, dissolve it with solvent, extract it, and obtain the test solution; b. Determine the test solution by high performance liquid chromatography to obtain the characteristic chromatogram of Tianlong or its preparation; The high performance liquid chromatography conditions are as follows: the chromatographic column is C18 column; acetonitrile is used as mobile phase A, and 0.1% acetic acid solution is used as mobile phase B for gradient elution; the gradient elution conditions are as follows:

2. The method of constructing a characteristic profile of Shendrughai or its preparation as claimed in claim 1, wherein: The preparation method of the test sample solution is as follows: take the test sample, accurately weigh it, place it in a conical flask with a plug, accurately add water, tightly plug, weigh, ultrasonically treat, cool, weigh again, make up the weight loss with water, shake well, filter, and take the filtrate to obtain the test solution.

3. The method of constructing a characteristic profile of Shendrugh or its preparation as claimed in claim 2, wherein: The ultrasonic treatment conditions are as follows: power 600W, frequency 40kHz, and treatment time 30 minutes.

4. The method of constructing a characteristic profile of Shendrugh or its preparation as claimed in claim 1, wherein: The flow rate of the mobile phase is 1.0ml per minute; the column temperature is 30°C; and the detection wavelength is 260nm; the theoretical plate number calculated according to the peak of pterin-6-carboxylic acid should not be less than 5000.

5. The method of constructing a characteristic profile of Shendrugh or its formulation as claimed in claim 1 wherein: It also comprises the preparation of reference solution, which comprises Tianlong control medicinal material, pterin-6-carboxylic acid control, hypoxanthine control, and thymine control. The preparation method of the reference solution of Tianlong control medicinal material is as follows: take the Tianlong control medicinal material, add water, ultrasonically treat, cool, shake well, filter, and take the filtrate as the reference solution of control medicinal material; The preparation method of the reference solution of pterin-6-carboxylic acid control, hypoxanthine control, and thymine control is as follows: take pterin-6-carboxylic acid control, hypoxanthine control, and thymine control, add water or ammonia solution to prepare the reference solution.

6. The method of constructing a characteristic pattern of a Tianlong or preparation thereof according to any one of claims 1-5, characterized in that: The characteristic chromatogram contains 8 characteristic peaks, and the relative retention times are as follows: Peak 1: 0.38, peak 2: 0.42, peak 3: 0.52, peak 4: 0.65, peak 5: 1.0, peak 6: 1.06, peak 7: 1.25, and peak 8: 1.45, with a relative retention time floating within ±10%. Peak 3 is hypoxanthine; peak 4 is thymine; and peak 5 is pterin-6-carboxylic acid. The Tianlong or its preparation comprises Tianlong medicinal material, decoction piece, standard decoction, and formula granule. It comprises the following steps:

7. The method of constructing a characteristic profile of Shendrugh or its formulation as claimed in claim 6, wherein: a. Weigh the sample to be detected; 8. The method of constructing a characteristic pattern of Shendrugs or its preparation according to any one of claims 1-7, characterized in that: b. Detect the characteristic peaks according to the characteristic chromatogram construction method of Tianlong or its preparation according to any one of claims 1-8; if the 8 characteristic peaks are completely consistent, it is the genuine Tianlong medicinal material.

9. A method for identifying confused products of Tianlong medicinal materials, characterized in that: The confused product of Tianlong medicinal material is Eupolia sinensis. ​ ​ 10. The method of claim 7, wherein the method is characterized by: ​

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