A fingerprint construction method and content testing method for a longan pulp medicinal preparation

Abstract

The present invention relates to the field of detection of traditional Chinese medicine preparations, and specifically provides a method for constructing a fingerprint of a longan pulp medicinal preparation and a method for testing its content. The method adopts a test sample solution for high performance liquid chromatography detection, uses octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, and an aqueous solution containing phosphoric acid as a mobile phase B, and adopts a specific elution procedure. The method shortens the detection time and significantly improves the separation effect of multiple effective ingredients, so that more characteristic peaks are included in the fingerprint, greatly enriches the spectral information, and realizes the effective separation of characteristic peaks including uridine, 5-hydroxymethylfurfural, uracil, and guanosine. The obtained fingerprint has obvious characteristics and can effectively and comprehensively detect the longan pulp medicinal preparation.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine detection, and in particular to a method for constructing a fingerprint of a longan pulp medicinal preparation and a method for testing its content. Background Art

[0002] Longan meat granules are obtained by extracting, concentrating, and granulating the traditional Chinese medicine (TCM) longan meat. Longan meat, the aril of the Sapindaceae plant Dimocarpus longan Lour., was first recorded in the "Shennong Bencao Jing." Li Shizhen of the Ming Dynasty said, "Lychee is the most valuable food, while longan is the best for health benefits." Longan meat is sweet, neutral, and non-toxic. It enters the heart and spleen meridians. Its properties are neither hot nor cold, and its peace of mind is valuable, contributing to the development of wisdom. Longan meat is rich in nutrients, with its main chemical components being sugars, nucleosides, and polypeptides. Modern pharmacological research has shown that longan meat has antioxidant, anti-aging, antibacterial, and anti-tumor effects. Longan meat is a pharmacopoeial TCM, and the Chinese Pharmacopoeia does not yet have established standards for its compositional analysis. Literature also describes the chemical components of longan meat. However, on the one hand, the content determination or identification of longan meat formula granules through the above-mentioned single component cannot detect and control its quality as a whole; on the other hand, the content determination of a single component combined with the identification of other components of longan meat formula granules is time-consuming and labor-intensive, making it difficult to be widely applied in production practice.

[0003] However, existing methods for controlling the quality of longan pulp are primarily focused on the raw material, not the pharmaceutical preparations. Consequently, existing methods for controlling the raw material are not applicable to pharmaceutical preparations. Therefore, establishing a comprehensive and rapid method for testing pharmaceutical preparations is crucial for comprehensive quality testing and overall quality control. Summary of the Invention

[0004] Therefore, the object of the present invention is to provide a method for constructing a fingerprint spectrum of a pharmaceutical preparation of longan meat and a content testing method. This method establishes a fingerprint spectrum of this variety based on the characteristics of the pharmaceutical preparation of longan meat, realizes effective separation of each characteristic peak, increases the number of characteristic peaks, and at the same time shortens the detection and analysis time, improves the separation effect, and can quickly and comprehensively detect the pharmaceutical preparation of longan meat.

[0005] To this end, the present invention provides a method for constructing a fingerprint of a pharmaceutical preparation of longan pulp, comprising the following steps:

[0006] Prepare test sample solution;

[0007] The test solution was detected by high performance liquid chromatography. The chromatographic conditions included using octadecylsilane bonded silica gel as the filler, methanol as the mobile phase A, and an aqueous solution containing phosphoric acid as the mobile phase B. Gradient elution was performed according to the following procedure:

[0008] 0-15min, the volume ratio of mobile phase A to mobile phase B is 0%:100%,

[0009] 15-50min, the volume ratio of mobile phase A to mobile phase B is 0%:100%→5%:95%,

[0010] 50-60 min, the volume ratio of mobile phase A to mobile phase B is 5%:95% → 8%:92%.

[0011] Furthermore, a chromatographic column with a specification of 250 mm × 4.6 mm and 5 μm is used; and / or the injection volume of the test solution is 5-20 μL; and / or the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.1-0.3%; and / or the detection wavelength is 260-264 nm, the column temperature is 20-35° C., and the flow rate is 0.5-1.0 mL / min.

[0012] Furthermore, the column temperature is 23-27°C.

[0013] Furthermore, the flow rate is 0.7-0.9 mL / min.

[0014] Furthermore, a Shimadzu GL science Inertil ODS-3 chromatographic column with a specification of 250 mm × 4.6 mm and 5 μm was used.

[0015] Furthermore, the preparation method of the test solution is: weighing the test sample, adding a solvent for extraction to obtain an extract, separating the solid and the liquid, and taking the liquid, which is the test solution.

[0016] The preparation method of the test solution also satisfies any one or more of the following AE:

[0017] A. The ratio of the mass of the test sample to the volume of the solvent is 0.2-1.0:10-50; the relationship between mass and volume is g / mL;

[0018] B. The extraction method is reflux extraction or ultrasonic extraction;

[0019] C. Extraction time is ≥10 min, preferably 15-60 min;

[0020] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0021] E. The solvent is selected from at least one of water, methanol and ethanol, preferably water or methanol aqueous solution.

[0022] Furthermore, the solvent is selected from a methanol aqueous solution with a volume percentage of 5-50%. The solvent is selected from a methanol aqueous solution with a volume percentage of 5-30%.

[0023] Illustratively, the method for preparing the test solution is: take the test sample, accurately add the solvent, seal it, weigh it, perform ultrasonic treatment, take it out, let it cool, weigh it again, make up the lost weight with the solvent, shake it well, filter it, and take the filtrate to obtain it.

[0024] Furthermore, the construction method further includes the step of preparing a reference substance solution by using at least one of uridine, 5-hydroxymethylfurfural, uracil, and guanosine as a reference substance and a solvent, and the step of detecting the reference substance solution by high performance liquid chromatography according to any of the above construction methods to obtain a reference substance spectrum; preferably, the solvent is selected from at least one of methanol, ethanol, and water; preferably, 1 mL of the reference substance solution contains 0.005 to 50 μg (for example, 1 to 50 μg) of each reference substance.

[0025] Furthermore, the fingerprint of the pharmaceutical preparation of longan pulp has 6 common characteristic peaks, peak 2 and peak 5 correspond to the retention times of uridine and 5-hydroxymethylfurfural reference substance peaks, respectively, the peak corresponding to the uridine reference substance peak is the S1 peak, and the relative retention times of peak 1, peak 3 to peak 4, peak 6 and S1 peak are within ±10% of the specified value, and the specified values ​​of peak 1, peak 3 to peak 4, and peak 6 are: 0.40, 1.18, 1.56, and 2.28, respectively.

[0026] The present invention also provides a method for testing the content of a longan pulp pharmaceutical preparation, comprising the following steps:

[0027] Prepare test sample solution;

[0028] preparing a uridine reference solution and / or a 5-hydroxymethylfurfural reference solution;

[0029] The test solution and the uridine reference solution and / or 5-hydroxymethylfurfural reference solution were detected by high performance liquid chromatography. The chromatographic conditions included using octadecylsilane bonded silica gel as the filler, methanol as the mobile phase A, and an aqueous solution containing phosphoric acid as the mobile phase B, and gradient elution according to the following procedure:

[0030] 0-15min, the volume ratio of mobile phase A to mobile phase B is 0%:100%,

[0031] 15-50min, the volume ratio of mobile phase A to mobile phase B is 0%:100%→5%:95%,

[0032] 50-60 min, the volume ratio of mobile phase A to mobile phase B is 5%:95% → 8%:92%.

[0033] Furthermore, a Shimadzu GL science Inertil ODS-3 chromatographic column with a specification of 250 mm × 4.6 mm and 5 μm is used; and / or the injection volume of the test solution is 5-20 μL; and / or the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.1-0.3%; and / or the detection wavelength is 260-264 nm, the column temperature is 20-35°C, and the flow rate is 0.5-1.0 mL / min.

[0034] Furthermore, the preparation method of the test solution is: weighing the test sample, adding a solvent for extraction, obtaining an extract, separating the solid and the liquid, and taking the liquid, which is the test solution. Preferably, the preparation method of the test solution also meets any one or more of the following AE:

[0035] A. The ratio of the mass of the test sample to the volume of the solvent is 0.3-0.7:10-50; the relationship between mass and volume is g / mL;

[0036] B. The extraction method is reflux extraction or ultrasonic extraction;

[0037] C. Extraction time is ≥10 min, preferably 15-60 min;

[0038] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0039] E. The solvent is selected from at least one of water, methanol and ethanol, preferably water or methanol aqueous solution.

[0040] Furthermore, the solvent used in the preparation of the uridine reference solution and / or the 5-hydroxymethylfurfural reference solution is selected from at least one of methanol, ethanol and water; and / or the concentration of the uridine reference solution is 0.03 to 50 μg / mL (e.g., 0.03 to 5 μg / mL); and / or the concentration of the 5-hydroxymethylfurfural reference solution is 0.005 to 50 μg / mL (e.g., 0.005 to 1 μg / mL).

[0041] The present invention also provides a method for detecting the quality of a pharmaceutical preparation of longan meat, comprising the steps of comparing the fingerprint of the longan meat product to be tested with a control fingerprint of the pharmaceutical preparation of longan meat and / or determining the content of the longan meat product to be tested by using any of the above-mentioned content testing methods for the pharmaceutical preparation of longan meat; the fingerprint of the longan meat product to be tested is constructed using the longan meat product to be tested according to any of the above-mentioned construction methods, and the control fingerprint of the pharmaceutical preparation of longan meat is selected from any one of the following (1)-(3):

[0042] (1) It has 6 common characteristic peaks, peak 2 and peak 5 correspond to the retention times of uridine and 5-hydroxymethylfurfural reference substance peaks, respectively. The peak corresponding to the uridine reference substance peak is the S1 peak. The relative retention times of peak 1, peak 3 to peak 4, and peak 6 to the S1 peak are within the range of ±10% of the specified values. The specified values ​​of peak 1, peak 3 to peak 4, and peak 6 are: 0.40, 1.18, 1.56, and 2.28, respectively.

[0043] (2) fingerprints of pharmaceutical preparations of longan pulp obtained by using a single batch or multiple batches of pharmaceutical preparations of longan pulp according to any of the above construction methods;

[0044] (3) The fingerprints of pharmaceutical preparations of multiple batches of longan pulp obtained according to any of the construction methods described above are used to prepare control fingerprints by using the average or median method.

[0045] Among them, the longan meat product to be tested is a pharmaceutical preparation of longan meat.

[0046] The similarity of the longan pulp formula granules to the control fingerprint is greater than 0.800.

[0047] The technical solution of the present invention has the following advantages:

[0048] 1. The method for constructing the fingerprint of the pharmaceutical preparation of longan meat provided by the present invention uses octadecylsilane bonded silica gel as a filler, methanol as mobile phase A, and an aqueous solution containing phosphoric acid as mobile phase B, and adopts a specific elution procedure. While shortening the detection time, it also significantly improves the separation effect of multiple effective ingredients, so that more characteristic peaks are included in the fingerprint, greatly enriching the spectral information, and realizing the effective separation of characteristic peaks including uridine, 5-hydroxymethylfurfural, uracil, and guanosine. The obtained fingerprint is obvious in characteristic, fully reflecting the integrity and characteristic of the pharmaceutical preparation of longan meat, and the baseline is stable and the peak shape is good, providing a basis for comprehensive detection of the quality of the pharmaceutical preparation of longan meat.

[0049] 2. The content testing method of the pharmaceutical preparation of longan pulp provided by the present invention uses octadecylsilane bonded silica gel as a filler, methanol as mobile phase A, and an aqueous solution containing phosphoric acid as mobile phase B, and adopts a specific elution procedure. It can simultaneously determine the contents of components such as uridine and 5-hydroxymethylfurfural, and the separation degree of each component is good. In addition, the constructed method has high precision, good stability and repeatability. Therefore, it can comprehensively and quickly detect the effective ingredients and their contents in the longan pulp formula granules.

[0050] 3. The method for constructing the fingerprint of the longan pulp pharmaceutical preparation provided by the present invention can select S-peak uridine as the internal reference peak in the fingerprint, and can determine the six common characteristic peaks of the longan pulp formula granules, and calculate the relative retention time of each common characteristic peak based on the S-peak uridine, which is beneficial to the comprehensive quality detection and overall quality control of the longan pulp formula granules, thereby helping to improve the safety and stable uniformity of the drug use.

[0051] 4. The quality detection method of the fingerprint of the pharmaceutical preparation of longan pulp provided by the present invention can simultaneously construct the fingerprint of the longan pulp to be tested and perform content determination. The method is simple and convenient for comprehensive control of the quality of longan pulp. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0053] Figure 1 The fingerprints of 15 batches of longan pulp formula granules obtained by high performance liquid chromatography in Example 1 of the present invention are as follows;

[0054] Figure 2 This is the control fingerprint generated from 15 batches of longan pulp formula granules in Example 1 of the present invention;

[0055] Figure 3 It is the positioning diagram of the control fingerprint and different reference substances in Example 1 of the present invention;

[0056] Figure 4 This is the reference medicinal material fingerprint of longan pulp in Example 1 of the present invention;

[0057] Figure 5 This is the spectrum of the negative control solution in the specificity experiment in Example 2 of the present invention;

[0058] Figure 6 This is the fingerprint of the longan pulp formula granules in the specific property experiment in Example 2 of the present invention;

[0059] Figure 7 This is the chromatogram at a column temperature of 20° C. in Example 3 of the present invention;

[0060] Figure 8 This is the chromatogram at a column temperature of 25° C. in Example 3 of the present invention;

[0061] Figure 9 This is the chromatogram at a column temperature of 30° C. in Example 3 of the present invention;

[0062] Figure 10 This is the chromatogram at a column temperature of 35° C. in Example 3 of the present invention;

[0063] Figure 11 This is the chromatogram at a flow rate of 0.5 ml / min in Example 4 of the present invention;

[0064] Figure 12 This is the chromatogram at a flow rate of 0.8 ml / min in Example 4 of the present invention;

[0065] Figure 13 This is the chromatogram at a flow rate of 1.0 ml / min in Example 4 of the present invention;

[0066] Figure 14 is the Shimadzu GL Sciences Inertsil ODS-3 chromatogram in Example 5 of the present invention;

[0067] Figure 15 is the Hanbon Hedera ODS-2 chromatogram in Example 5 of the present invention;

[0068] Figure 16 is the OSAKA SODA CAPCELL PAK AQ C18 chromatogram in Example 5 of the present invention;

[0069] Figure 17 is the chromatogram of the Waters Arc chromatograph in Example 6 of the present invention;

[0070] Figure 18 is a chromatogram of the Dionex U3000 chromatograph in Example 6 of the present invention;

[0071] Figure 19 is a chromatogram of the uridine reference substance in Example 7 of the present invention;

[0072] Figure 20 This is a chromatogram of the 5-hydroxymethylfurfural reference substance in Example 7 of the present invention;

[0073] Figure 21 This is a chromatogram of the longan pulp formula granule test sample in Example 7 of the present invention;

[0074] Figure 22 This is a linear relationship diagram of different concentrations of uridine reference substances in Example 9 of the present invention;

[0075] Figure 23 This is a linear relationship diagram of different concentrations of 5-hydroxymethylfurfural reference substances in Example 9 of the present invention;

[0076] Figure 24 This is the chromatogram obtained by using gradient elution procedure 1 in Comparative Examples 1 to 3 of the present invention;

[0077] Figure 25 This is the chromatogram obtained by using gradient elution procedure 2 in Comparative Examples 1 to 3 of the present invention;

[0078] Figure 26 This is the chromatogram obtained by using gradient elution procedure 3 in Comparative Examples 1 to 3 of the present invention;

[0079] Figure 27 This is a chromatogram obtained using acetonitrile as mobile phase A in Comparative Example 4 of the present invention;

[0080] Figure 28 This is the chromatogram obtained in Comparative Examples 5 to 7 of the present invention using 0.2% formic acid as mobile phase B;

[0081] Figure 29 This is the chromatogram obtained in Comparative Examples 5 to 7 of the present invention using 0.2% acetic acid as mobile phase B;

[0082] Figure 30 This is the chromatogram obtained using water as mobile phase B in Comparative Examples 5 to 7 of the present invention. DETAILED DESCRIPTION

[0083] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0084] The instruments and reagents used in the present invention are as follows:

[0085] HPLC 1: Thermo Fisher U3000 chromatography system, including LPG-3400A quaternary pump, WPS-3000TSL autosampler, PDA diode array detector, and chromatography workstation; HPLC 2: Waters Arc chromatography system, including quaternary gradient infusion pump, 120-position high-performance autosampler, imported column oven, Waters 2998 diode array UV detector, and Empower chromatography management system; 1 / 10,000 electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., FA1004); 1 / 100,000 electronic balance (Sartorius Scientific Instrument Co., Ltd., SQP); Ultrasonic cleaning machine: WB400US (Shanghai Wangbiao Instrument Co., Ltd.); Chromatographic columns: (1) Shimadzu GL Sciences Inertsil ODS-3 (4.6 mm × 250 mm, 5 μm); (2) Hanbon Hedera ODS-2 (4.6 mm × 250 mm, 5 μm); (3) OSAKA SODA CAPCELL PAK AQ C18 (4.6 mm × 250 mm, 5 μm). Methanol and acetonitrile were chromatographically pure (Merck); phosphoric acid (chromatographic grade, 85-90%, Aladdin); glacial acetic acid (HPLC, >99.9%, Aladdin); formic acid (chromatographic grade, ≥98%, Aladdin); water (Milli-Q). Uridine reference substance (batch number: 110887-202104, purchased from the China Food and Drug Administration); 5-hydroxymethylfurfural reference substance (batch number: 111626-202316, purchased from the China Food and Drug Administration); longan meat reference medicinal material (batch number: 121613-201202, purchased from the China Food and Drug Administration); longan meat formula granules (1902002W, 190 : 4002S, 1901001S, 1903001W, 1903001W, 1910002S, 1912001W, 2004001S, 2011002W, 2105001S, 2108002S, 2112002S, 2203001W, 2209003S, 2212001S); negative particles (lot number: 2104001Y).

[0086] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0087] Example 1

[0088] A method for determining the content of a longan pulp pharmaceutical preparation and detecting its fingerprint spectrum comprises:

[0089] (1) Preparation of test solution: Take approximately 0.5 g of the powder of the pharmaceutical preparation of longan pulp, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 10% methanol, stopper it tightly, weigh it, and ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes. Remove it, let it cool, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate. The pharmaceutical preparation of longan pulp described in the present invention is prepared by the following method:

[0090] Take longan pulp, heat and reflux extract at least once, add 8 to 14 times the weight of water each time for at least 0.5 hours, filter, combine the filtrates, concentrate the filtrates to a relative density of 1.05 to 1.10 g / mL at 60°C, add conventional excipients, and prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations or injection preparations according to conventional processes. The pharmaceutically acceptable excipients include fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include: polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavorings include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include: PEG6000, PEG4000, insect wax, etc.

[0091] The test sample in this embodiment is longan pulp formula granules. The specific preparation method of the longan pulp formula granules is as follows: take longan pulp, heat and reflux extraction twice, add 8 times the weight of water to soak for 30 minutes for the first time, heat and reflux extraction for 0.5 hours, filter, add 6 times the weight of water to extract for 0.5 hours for the second time, filter, combine the filtrate, concentrate the filtrate to a relative density of 1.05 g / mL at 60°C, spray dry, add maltodextrin as an auxiliary material to the dry powder, mix well, and then dry granulate to make granules.

[0092] (2) Preparation of reference solution: Take 1 g of longan pulp as a reference medicinal material, place it in a stoppered conical flask, add 25 mL of water, heat and reflux for 45 minutes, filter, evaporate the filtrate to dryness, add 10 mL of 10% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Separately, take an appropriate amount of uridine reference substance, accurately weigh it, add 10% methanol to make a solution containing 30 μg of uridine per 1 mL, and obtain the reference solution I. Take an appropriate amount of 5-hydroxymethylfurfural reference substance, accurately weigh it, add methanol to make a solution containing 5 μg of 5-hydroxymethylfurfural per 1 mL, and obtain the reference solution II. Take an appropriate amount of guanosine reference substance, accurately weigh it, add 10% methanol to make a mixed solution containing 30 μg of guanosine per 1 mL, and obtain the reference solution III. Then take an appropriate amount of uracil reference substance, accurately weigh it, and add 10% methanol to make a mixed solution containing 30 μg of uracil per 1 mL, which is used as the reference substance solution IV.

[0093] (3) High performance liquid chromatography detection

[0094] Test the test and reference solutions by high performance liquid chromatography (HPLC) using the following conditions: octadecylsilane bonded silica gel as the filler; methanol as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; detection wavelength at 262 nm, column temperature at 25°C, flow rate at 0.8 mL / min. The number of theoretical plates, calculated based on the uridine peak, should be no less than 2500.

[0095] Table 1 Gradient elution program

[0096]

[0097] The common pattern of the fingerprint of longan pulp formula particles was established according to the above method.

[0098] Fifteen batches of longan pulp formula granule samples were taken respectively to obtain the longan pulp formula granule fingerprints according to the above method, as shown in FIG. Figure 1 As shown. Figure 1 The batch numbers of S1 to S15 are 1902002W, 1904002S, 1901001S, 1903001W, 1903001W, 1910002S, 1912001W, 2004001S, 2011002W, 2105001S, 2108002S, 2112002S, 2203001W, 2209003S, and 2212001S. R(6) is the control fingerprint of the longan pulp formula granules.

[0099] The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate the reference fingerprints, such as Figure 2 As shown. Figure 2 The control fingerprint shown can be used to analyze and compare the test results of the formula granule fingerprint for quality control of the formula granule.

[0100] pass Figure 1 、 Figure 2 and Figure 3 It can be seen that there are 6 characteristic peaks in the control fingerprint. Among them, peak 2 and peak 5 correspond to the retention time of the uridine and 5-hydroxymethylfurfural reference peaks; the peak corresponding to the uridine reference peak is the S peak, and the relative retention times of each characteristic peak and the S peak are: 0.40 (peak 1), 1.18 (peak 3), 1.56 (peak 4), and 2.28 (peak 6). See Table 2. Figure 4 As shown in the figure, the fingerprint of the control medicinal material presents 6 characteristic peaks, the peak corresponding to the uridine reference peak is the S peak, and the relative retention times of each characteristic peak and the S peak are: 0.39 (peak 1), 1.19 (peak 3), 1.56 (peak 4), and 2.28 (peak 6).

[0101] The fingerprints of 15 batches of longan pulp formula granules were used to calculate the relative retention times of peak 1, peak 3 to peak 4, peak 6 and peak S1. The relative retention times were within the range of ±10% of the specified values, which were: 0.40 (peak 1), 1.18 (peak 3), 1.56 (peak 4), 2.28 (peak 6). The relative peak area between peak 5 and peak S was no more than 2.0. The similarity of the fingerprints of 15 batches of longan pulp formula granules was greater than 0.80. The results are shown in Tables 3 to 6. The reference samples of different characteristic peaks are located as shown in Tables 3 to 6. Figure 3 As shown, peak 1 is uracil; peak 2 (S peak) is uridine; peak 4 is guanosine; and peak 5 is 5-hydroxymethylfurfural.

[0102] Table 2 Relative retention time of common patterns in fingerprint of longan pulp formula granules

[0103]

[0104] Table 3 Common pattern matching data of longan pulp formula particles

[0105]

[0106] Table 4 Relative retention time results of fingerprint determination of 15 batches of longan pulp formula granules

[0107]

[0108]

[0109] Table 5 Relative peak area results of characteristic fingerprints of 15 batches of longan pulp granules

[0110]

[0111]

[0112] Table 6 Similarity results of fingerprint spectra of 15 batches of longan pulp granules

[0113]

[0114] Table 7 Content determination results of 15 batches of longan pulp granules

[0115]

[0116] The construction method described in this example effectively obtains a fingerprint with good resolution of each characteristic peak, allowing for simultaneous determination of the uridine and 5-hydroxymethylfurfural contents. Furthermore, by selecting the S-peak uridine as the internal reference peak in the fingerprint, the relative retention times of peaks 1, 3, and 6, the common characteristic peaks of the longan pulp granules, can be determined. This allows for comprehensive and rapid testing of the longan pulp granules, facilitating comprehensive quality testing and overall quality control of the granules, thereby contributing to improved safety, stability, and uniformity of the drug.

[0117] Example 2 Methodology Verification

[0118] 1. Precision

[0119] 1.1 Precision experiment

[0120] The same sample solution prepared by the method of Example 1 (longan pulp formula granules with batch number 2203001W) was injected six times according to the chromatographic conditions of item (3) of Example 1. The relative retention times of the six common peaks were measured. The RSD values ​​of the relative retention times of the peaks were 0.05%, 0%, 0.01%, 0.02%, 0.02%, and 0.03%, respectively. The RSD values ​​of the relative peak areas were 0.34%, 0%, 0.20%, 1.13%, 0.36%, and 1.73%, respectively. The RSD of the relative retention times of the characteristic peaks was less than 2.0%, indicating good instrument precision.

[0121] Table 8 Precision test relative retention time results

[0122]

[0123] 1.2 Repeatability Experiment

[0124] Six portions of the same batch of test sample (longan pulp formula granules with batch number 2203001W) were taken, and the relative retention times of the six common peaks were respectively determined according to the method of Example 1. The RSD values ​​of the relative retention times of the peaks were 0.34%, 0%, 0.20%, 1.13%, 0.36%, and 1.73%, respectively. The RSD values ​​of the relative peak areas were 0.44%, 0%, 0.34%, 0.96%, 0.07%, and 0.30%, respectively. The RSD of the relative retention time of the characteristic peaks was less than 2.0%. The results showed that the method had good repeatability.

[0125] Table 9 Relative retention time results of repeatability test

[0126]

[0127]

[0128] 2. Specificity experiment

[0129] The test sample was longan pulp formula granules of batch number 2203001W. The test solution was prepared according to the method of Example 1. 0.4 g of maltodextrin was weighed to replace the test sample. A negative control solution was prepared according to the same method as the test solution. The test was performed according to the method of Example 1. The test results were as follows: Figure 5 and Figure 6 As shown, the results showed that the negative control solution had no interference.

[0130] 3. Stability test

[0131] The same batch number of the test sample (longan pulp formula granules with batch number 2203001W) was taken and operated according to the construction method of Example 1. The samples were injected at 0h, 2h, 4h, 8h, 12h, and 24h, respectively, and the relative retention times of the 6 common peaks were determined. The RSD values ​​of the relative retention times of the peaks were 0.12%, 0%, 0.06%, 0.04%, 0.04%, and 0.05%, respectively. The RSD values ​​of the relative peak areas were 0.75%, 0%, 1.11%, 0.89%, 0.59%, and 3.04%, respectively. The RSD of the relative retention time of the characteristic peak was less than 2.0%. The results showed that the test solution was stable within 24h and met the measurement requirements.

[0132] Table 10 Relative retention time results of stability test

[0133]

[0134] 4. Intermediate precision (personnel)

[0135] Three experimenters prepared and measured the test solution of longan pulp formula granules (2203001W) according to the method of Example 1. The RSD values ​​of the relative retention time of each peak were 0.09%, 0%, 0.01%, 0.04%, 0.02%, and 0.03%, respectively. The RSD values ​​of the relative peak areas were 0.90%, 0%, 0.53%, 1.24%, 0.97%, and 0.32%, respectively. The RSD of the relative retention time of the six characteristic peaks should be less than 2.0%. The results show that the intermediate precision is good.

[0136] Table 11 Intermediate precision (personnel) relative retention time

[0137]

[0138]

[0139] 5. Different flow rates

[0140] The same sample solution (longan meat formula granules of batch number 2203001W) prepared by the method of Example 1 was taken to compare the effects of different flow rates on the durability of the fingerprint of longan meat formula granules (2203001W). The flow rates were set to 0.7 ml / min, 0.8 ml / min, and 0.9 ml / min, respectively. The rest of the chromatographic conditions were based on the conditions under item (3) of Example 1. The effects of different flow rates on the separation effect of the longan meat formula granules were observed. The results showed that with slight fluctuations in flow rate, the separation effect of each chromatographic peak was good and the system adaptability parameters were relatively close. The relative retention time RSD values ​​of each peak were 0.79%, 0%, 0.38%, 1.77%, 1.82%, and 2.13%, respectively, all less than 3%, indicating that the durability of different flow rates was good.

[0141] Table 12 Relative retention time results at different flow rates

[0142]

[0143] 6. Different column temperatures

[0144] The same sample solution (longan pulp formula granules of batch number 2203001W) prepared by the method of Example 1 was taken to compare the effect of different column temperatures on the durability of the fingerprint of longan pulp formula granules (2203001W). The column temperature was set at 23°C, 25°C, and 27°C, and the rest of the chromatographic conditions were as in item (3) of Example 1. The effect of different column temperatures on the separation effect of the longan pulp formula granules was observed. The results showed that with the fluctuation of column temperature, the separation effect of each chromatographic peak was good and the system adaptability parameters were relatively close. The relative retention time RSD values ​​of each peak were respectively: 1.48%, 0%, 1.82%, 0.18%, 1.55%, and 1.29%, all less than 2%, indicating that the durability of different column temperatures was good.

[0145] Table 13 Relative retention time results at different column temperatures

[0146]

[0147] 7. Different chromatographic columns

[0148] The same sample solution (longan pulp formula granules, batch number 2203001W) prepared by the method of Example 1 was used to compare the effects of different chromatographic columns (column 1: Shimadzu GL Sciences Inertsil ODS-3; column 2: Hanbon Hedera ODS-2; column 3: OSAKA SODA CAPCELL PAK AQ C18) on the separation of the fingerprint of the longan pulp formula granules. The remaining chromatographic conditions were the same as those in item (3) of Example 1. The relative retention time RSD values ​​of the peaks were 4.17%, 0%, 3.73%, 4.69%, 3.53%, and 3.50%, respectively, all less than 5%, indicating that the different chromatographic columns had good durability.

[0149] Table 14 Comparison of relative retention time results of different brands of chromatographic columns

[0150]

[0151] 8. Different chromatographs

[0152] The same longan pulp formula granules (2203001W) were used as a test sample. The test sample solution was prepared according to the method of Example 1. The fingerprints were measured on Dionex and Waters HPLC, respectively. The spectra and relative retention time results measured by different instruments were compared. The rest of the chromatographic conditions were based on the chromatographic conditions under item (3) of Example 1. Comparison of the fingerprints measured by the two HPLCs revealed that the chromatographic information presented by the two different brands of HPLCs was relatively complete, and none of the characteristic peaks were missing. The relative retention time RSD values ​​of the peaks were 1.01%, 0%, 0.41%, 0.19%, 0.03%, and 0.28%, respectively, indicating that the instrument durability of different models of HPLCs for this method was good.

[0153] Table 15 Relative retention time of instrument durability investigation

[0154]

[0155] Example 3

[0156] The purpose of this example is to investigate the effect of different column temperatures on the detection of longan pulp formula granules test solution. The test solution was prepared using longan pulp formula granules according to the method of Example 1, and the column temperature was set to 20°C, 25°C, 30°C, and 35°C respectively. The other chromatographic conditions were the same as in Example 1. The test results are shown in the following table and Figure 7-10 As shown in the table and accompanying figure, the temperature range of 20-35°C is effective for the separation of multiple active ingredients in longan pulp granules, with a stable baseline and good peak shape.

[0157] Table 16 Results of investigation at different column temperatures

[0158]

[0159]

[0160] Example 4

[0161] The purpose of this example is to investigate the effect of different flow rates on the detection of longan pulp formula particles. The test solution is tested with the same batch of test solution as in Example 3. The flow rates are set to 0.5 ml / min and 1.0 ml / min respectively. The other chromatographic conditions are the same as in Example 1. The test results are compared with those in Example 3 with a column temperature of 25°C and a flow rate of 0.8 ml / min. The test results are shown in the following table and Figure 11-13 As shown in the figure, at a flow rate of 0.5 mL / min to 1.0 mL / min, separation of more than eight components in the longan pulp granules was achieved with a stable baseline and good peak shape. However, at flow rates of 0.5 mL / min and 1.0 mL / min, the separation of all components in the longan pulp granules was relatively poor, with a relatively small number of characteristic peaks. At a flow rate of 0.80 mL / min, separation of all components in the longan pulp granules was excellent, with a greater amount of peak information. Therefore, a flow rate of 0.80 mL / min is preferred.

[0162] Table 17 Investigation results at different flow rates

[0163]

[0164]

[0165] Example 5

[0166] This example investigates the effects of different chromatographic columns on the detection of longan pulp granules. A test solution was prepared using the longan pulp granules according to the method of Example 1. The chromatographic conditions differed from those in Example 1 in that different chromatographic columns were used. Column 1: Shimadzu GL Sciences Inertsil ODS-3; Column 2: Hanbon Hedera ODS-2; and Column 3: OSAKA SODA CAPCELL PAK AQ C18 were used. System suitability parameters for the different columns are shown in Table 18.

[0167] Table 18 Results of investigation on different chromatographic columns

[0168]

[0169]

[0170] Through the above table and Figure 14-16 It can be seen that: using the number of detected chromatographic peaks and the system suitability parameters of each chromatographic peak as evaluation indicators, different chromatographic columns can achieve effective separation of more than 8 components, with a stable baseline and good peak shape. Among them, the Shimadzu GL Sciences Inertsil ODS-3 (4.6mm×250mm, 5μm) chromatographic column has the best separation effect.

[0171] Example 6

[0172] This example investigates the effects of different chromatographic instruments on the analysis of longan pulp granules. A test solution was prepared using the longan pulp granules according to the method of Example 1. The difference from Example 1 lies in the different chromatographic instruments. The results were investigated using chromatographic instrument 1: Waters Arc; and chromatographic column 2: Thermo Fisher U3000. The system suitability parameters for the different chromatographic columns are shown in Table 19.

[0173] Table 19 Results of investigations with different instruments

[0174]

[0175] Through the above table and Figure 17 and 18 It can be seen that: using the number of detected chromatographic peaks and the system suitability parameters of each chromatographic peak as the selection indicators, different chromatographs can achieve effective separation of more than 10 components with a stable baseline and good peak shape.

[0176] Example 7

[0177] A method for determining the content of a longan pulp pharmaceutical preparation comprises:

[0178] (1) Preparation of test solution: Same as in Example 1, item (1).

[0179] (2) Preparation of Reference Solution: Take an appropriate amount of uridine reference substance, accurately weigh it, and add 10% methanol to prepare a mixed solution containing 30 μg of uridine per 1 mL. This is referred to as reference solution I. Also, take an appropriate amount of 5-hydroxymethylfurfural reference substance, accurately weigh it, and add methanol to prepare a solution containing 5 μg of 5-hydroxymethylfurfural per 1 mL. This is referred to as reference solution II.

[0180] (3) High performance liquid chromatography detection: Same as item (2) of Example 1.

[0181] The results are shown in the table below and Figure 19-21 shown.

[0182] Table 20 System adaptability parameters

[0183]

[0184] Example 8

[0185] The difference between this embodiment and embodiment 7 is that the sampling amount is different, and the effect of the sampling amount of the longan pulp formula granules on the extraction effect is investigated. The remaining processes and conditions are the same as those in embodiment 8. Specifically, an appropriate amount of this product is taken and ground into powder. About 0.2 g, 0.5 g, and 1.0 g are taken and accurately weighed. The mixture is placed in a stoppered conical flask, and 10 mL of a 10% methanol solution is accurately added. The flask is sealed and weighed. The mixture is ultrasonically treated (power 250 W, frequency 40 kHz) for 30 minutes. The mixture is allowed to cool and weighed again. The lost weight is supplemented with 10% methanol solution. The mixture is shaken and filtered. The filtrate is obtained.

[0186] Table 21 Content determination results of different sampling amounts

[0187]

[0188] The results show that the contents of index components in different sampling volumes are close, indicating that the extraction effects of different sampling volumes are similar.

[0189] Example 9 Methodology Verification

[0190] 1. Linear relationship investigation

[0191] Take an appropriate amount of uridine reference substance and add 10% methanol to prepare solutions containing 3.112μg, 1.556μg, 0.778μg, 0.3112μg, 0.1556μg, and 0.03112μg per 1mL. Accurately pipette 10μL of the above uridine reference substance solutions of different concentrations into the liquid chromatograph, measure the peak area, and draw a standard curve with the uridine injection amount as the horizontal axis and the peak area integral value as the vertical axis. The results are shown in the table below and Figure 22 , the regression equation is: y = 38.81x + 1.3325, R 2=0.9993. The experimental results showed that the linear relationship was good in the range of uridine injection amount from 0.03112μg to 3.112μg.

[0192] Take an appropriate amount of 5-hydroxymethylfurfural reference substance and add 10% methanol to prepare solutions containing 0.5433μg, 0.2717μg, 0.1358μg, 0.05433μg, 0.02717μg, and 0.005433μg per 1mL respectively. Accurately draw 10μL of the above 5-hydroxymethylfurfural reference substance solutions of different concentrations and inject them into the liquid chromatograph to measure the peak area. Use the injection amount of 5-hydroxymethylfurfural as the horizontal axis and the peak area integral value as the vertical axis to draw a standard curve. The results are shown in the table below and Figure 23 , the regression equation is: y = 218.52x - 1.8072, R 2 =0.999. The experimental results show that the linear relationship is good in the injection amount range of 5-hydroxymethylfurfural from 0.005433 μg to 0.5433 μg.

[0193] Table 22 Results of investigation on linear relationship of different concentrations of uridine

[0194]

[0195] Table 23 Results of linear relationship investigation of different concentrations of 5-hydroxymethylfurfural

[0196]

[0197] 2. Precision test

[0198] (1) Instrument precision test

[0199] The same longan pulp formula granule test solution (batch number: 2203001W) was taken and the sample was repeated 6 times according to the chromatographic conditions described in Example 7. The content and RSD values ​​of uridine and 5-hydroxymethylfurfural were determined. The RSD values ​​of the two components were 1.3% and 1.1%, respectively, both less than 2%, indicating good instrument precision.

[0200] (2) Investigation of the intermediate precision of different personnel

[0201] The same batch of longan pulp formula granules (batch number: 2203001W) was independently operated by experimenters A, B, and C, and processed according to the test sample preparation method in Example 8. The contents of uridine and 5-hydroxymethylfurfural were determined, and the RSD values ​​of each component were calculated. The RSD values ​​of the two components were 0.15% to 1.35%, respectively, both less than 2%. The results showed that this method had good precision among different operators.

[0202] (3) Repeatability study

[0203] Six portions of longan pulp granules (batch number: 2203001W) were prepared according to the preparation method of the test solution. The contents were determined according to the chromatographic conditions described in Example 8 and the RSD% of the content results was calculated. The RSD of uridine content was 0.84%, and the RSD of 5-hydroxymethylfurfural content was 0.85%, both less than 2%, indicating that the method had good reproducibility.

[0204] (4) Accuracy assessment

[0205] In the sample addition recovery test, approximately 0.25 g of 9 samples of longan pulp formula granules (batch number 2203001W) (the accompanying sample 5-hydroxymethylfurfural content was 0.301 mg / g) were accurately weighed and placed in stoppered conical flasks in groups of 3. 10 ml of 5-hydroxymethylfurfural reference solution (mass concentrations were 0.0331 mg / g, 0.0662 mg / g, and 0.0992 mg / g, respectively) were accurately added to each flask. The samples were processed according to the test sample preparation method of Example 8, and the recovery rate was calculated using the accompanying sample content. The recovery rate results are shown in the table below. The average recovery rate of 5-hydroxymethylfurfural was 100.1%, and the RSD values ​​were 0.73%, respectively, indicating that the method has good accuracy.

[0206] Approximately 0.25 g of nine samples of longan pulp formula granules (batch number 2203001W) (uridine content of the accompanying sample was 0.263 mg / g) were accurately weighed and placed in stoppered conical flasks in groups of three. 10 ml of uridine reference solution (mass concentrations of 0.00364 mg / g, 0.00729 mg / g, and 0.01093 mg / g, respectively) was accurately added to each sample. The samples were processed according to the test sample preparation method of Example 8. The recovery rate was calculated using the accompanying sample content. The recovery results are shown in the table below. The average uridine recovery rate was 99.8%, and the RSD value was 0.69%, indicating good accuracy of the method.

[0207] Table 24 Uridine accuracy test results

[0208]

[0209] Table 25 5-Hydroxymethylfurfural accuracy test results

[0210]

[0211]

[0212] 3. Durability inspection

[0213] (1) Solution stability

[0214] The same longan pulp formula granule test solution (batch number: 2203001W) was taken and sampled at 0, 2, 4, 8, 12, and 24 hours after preparation. The peak areas of uridine and 5-hydroxymethylfurfural were determined according to the chromatographic conditions described in Example 7, and the RSD values ​​of each component were calculated. The RSD values ​​of the two components were 1.3% and 0.6%, respectively, both less than 2%. The experimental results showed that the test solution was basically stable within 24 hours and could meet the measurement needs.

[0215] (2) Durability study at different flow rates

[0216] Longan pulp formula granules (batch number: 2203001W) were taken, accurately weighed, and processed according to the test sample preparation method of Example 8. The tests were carried out at different flow rates of 0.19 ml / min, 0.20 ml / min, and 0.21 ml / min, respectively. The rest were determined according to the chromatographic conditions described in Example 7 to determine the contents of uridine and 5-hydroxymethylfurfural. The RSD values ​​were calculated. The RSD values ​​of the two components were between 0.77% and 0.37%, respectively, both less than 2%. The results showed that different flow rates had little effect on the content of the index components and had good stability.

[0217] (3) Durability study at different column temperatures

[0218] Take longan pulp formula granules (batch number: 2203001W), accurately weighed, and processed according to the test sample preparation method of Example 8. The test was carried out at different flow rates of 23°C, 25°C, and 27°C, and the rest were determined according to the chromatographic conditions described in Example 7. The contents of uridine and 5-hydroxymethylfurfural were calculated, and the RSD values ​​of the two components were 0.64% and 0.44%, respectively, both less than 2%. The results show that different injection temperatures have little effect on the content of the index components and have good stability.

[0219] (4) Durability study of different chromatographic columns

[0220] Take longan pulp formula granules (batch number: 2203001W), accurately weighed, and processed according to the test sample preparation method in Example 8. The tests were carried out under different models of chromatographic columns of the same brand. The rest were determined according to the chromatographic conditions described in Example 7 to determine the contents of uridine and 5-hydroxymethylfurfural. The RSD values ​​were calculated. The RSD values ​​of the two components were between 0.3% and 1.4%, respectively, both less than 2%. The results showed that different chromatographic columns had little effect on the content of the index components.

[0221] (5) Durability inspection of different instruments

[0222] Take longan pulp formula granules (batch number: 2203001W), accurately weighed, processed according to the test sample preparation method in Example 8, and tested with different instruments. The contents of uridine and 5-hydroxymethylfurfural were determined according to the chromatographic conditions described in Example 7, and the RSD values ​​were calculated. The RSD values ​​of the two components were 1.4% and 1.2%, respectively, both less than 2%. The results show that different instruments have little effect on the content of the index components and have good stability.

[0223] Comparative Examples 1 to 3

[0224] The difference between the chromatographic conditions of Comparative Examples 1 to 3 and Example 3 at a column temperature of 25° C. is that the gradient elution program is different. The test solution is tested using the test solution from the same batch as Example 3. The gradient elution programs 1 to 3 of Comparative Examples 1 to 3 are shown in the following table:

[0225] Table 26 Gradient elution program 1

[0226]

[0227] Table 27 Gradient elution program 2

[0228]

[0229] Table 28 Gradient elution program 3

[0230]

[0231]

[0232] Table 29 Results of investigations at different gradients

[0233]

[0234] The fingerprint obtained by detection is as follows Figures 24-26 As shown, compared with the fingerprints obtained in each embodiment, the gradient conditions 1, 2, and 3 have less peak information and poorer separation.

[0235] Comparative Example 4

[0236] The difference between the chromatographic conditions of Comparative Example 4 and Example 3 at a column temperature of 25°C is that (3) the mobile phase A in the HPLC detection is different. Acetonitrile is used instead of methanol in this comparative example, and the test solution is tested using the test solution of the same batch as that of Example 3. The results are shown in the following table:

[0237] Table 30 Organic phase investigation results

[0238]

[0239] The fingerprint obtained by detection is as follows Figure 27As shown, compared with the fingerprints obtained in each embodiment, the peak information obtained by using acetonitrile-0.2% phosphoric acid is less and the separation is poorer.

[0240] Comparative Examples 5 to 7

[0241] The difference between the chromatographic conditions of Comparative Examples 5 to 7 and Example 3 at a column temperature of 25°C is that (3) the types of acids in the mobile phase in the HPLC detection are different. In Comparative Examples 5 to 7, 0.2% formic acid, 0.2% acetic acid, and water are used instead of 0.2% phosphoric acid as mobile phase B, respectively. The test solution is tested using the test solution from the same batch as in Example 3. The results are shown in the following table:

[0242] Table 31 Investigation results of different acid concentrations

[0243]

[0244] The fingerprint obtained by detection is as follows Figures 28-30 As shown, compared with the fingerprints obtained in each embodiment, the peak information obtained by using 0.2% formic acid, 0.2% acetic acid and water as mobile phase B is less and the separation is poorer.

[0245] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint of a pharmaceutical preparation of longan pulp, characterized in that: The steps include: Prepare a test solution; the test solution is prepared by weighing the test sample, adding a solvent to extract, obtaining an extract, separating the solid and the liquid, and taking the liquid, which is the test solution, wherein the solvent is selected from at least one of water, methanol and ethanol; The test solution and the reference solution were detected by high performance liquid chromatography. The chromatographic conditions included using octadecylsilane bonded silica gel as the filler, methanol as the mobile phase A, and an aqueous solution containing phosphoric acid as the mobile phase B. Gradient elution was performed according to the following procedure: 0-15min, the volume ratio of mobile phase A to mobile phase B is 0%:100%, 15-50min, the volume ratio of mobile phase A to mobile phase B is 0%:100%→5%:95%, The method comprises the following steps: a) performing a 50-60 min reaction, wherein the volume ratio of mobile phase A to mobile phase B is 5%:95%→8%:92%; b) performing a detection wavelength of 260-264 nm, and b) performing a 0.1-0.3% volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid; and c) performing a 0.7-0.9 mL / min flow rate. The method comprises the following steps: using uridine, 5-hydroxymethylfurfural, uracil, and guanosine as reference substances and adding a solvent to prepare the reference substance solution.

2. The method for constructing the fingerprint of the pharmaceutical preparation of longan pulp according to claim 1, characterized in that, A chromatographic column with a specification of 250 mm × 4.6 mm and 5 μm is used; and / or the injection volume of the test solution is 5-20 μL; and / or the column temperature is 20-35°C.

3. The method for constructing the fingerprint of the pharmaceutical preparation of longan pulp according to claim 1, characterized in that, The preparation method of the test solution also satisfies any one or more of the following AE: A. The ratio of the mass of the test sample to the volume of the solvent is 0.2-1.0:10-50; the relationship between mass and volume is g / mL; B. The extraction method is reflux extraction or ultrasonic extraction; C. Extraction time is ≥10 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration; E. The solvent is water or methanol aqueous solution.

4. The method for constructing the fingerprint of the pharmaceutical preparation of longan pulp according to claim 3, characterized in that: In the preparation method of the test solution, the extraction time is 15-60 minutes.

5. The method for constructing the fingerprint of the pharmaceutical preparation of longan pulp according to claim 1, characterized in that: The solvent used in the preparation process of the reference substance solution is selected from at least one of methanol, ethanol and water; and / or, 1 mL of the reference substance solution contains 0.005-50 μg of each reference substance.

6. The method for constructing the fingerprint of the pharmaceutical preparation of longan pulp according to any one of claims 1 to 5, characterized in that: The fingerprint of the pharmaceutical preparation of longan pulp has 6 common characteristic peaks, peak 2 and peak 5 correspond to the retention times of uridine and 5-hydroxymethylfurfural reference substance peaks, respectively, the peak corresponding to the uridine reference substance peak is the S1 peak, the relative retention times of peak 1, peak 3 to peak 4, peak 6 and S1 peak are within the range of ±10% of the specified value, and the specified values ​​of peak 1, peak 3 to peak 4, and peak 6 are: 0.40、1.18、1.56、2.28。 7. The method for constructing the fingerprint of the pharmaceutical preparation of longan pulp according to claim 6, characterized in that: The relative peak area between Peak 5 and Peak S is not greater than 2.

0.

8. A method for testing the content of a longan pulp pharmaceutical preparation, characterized in that: The steps include: Prepare a test solution; the test solution is prepared by weighing the test sample, adding a solvent to extract, obtaining an extract, separating the solid and the liquid, and taking the liquid, which is the test solution, wherein the solvent is selected from at least one of water, methanol and ethanol; Prepare uridine reference solution and 5-hydroxymethylfurfural reference solution; The test solution, uridine reference solution, and 5-hydroxymethylfurfural reference solution were detected by high performance liquid chromatography. The chromatographic conditions included using octadecylsilane bonded silica gel as a filler, a detection wavelength of 260-264 nm, methanol as mobile phase A, and an aqueous solution containing phosphoric acid as mobile phase B, wherein the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid was 0.1-0.3%. Gradient elution was performed according to the following procedure: 0-15min, the volume ratio of mobile phase A to mobile phase B is 0%:100%, 15-50min, the volume ratio of mobile phase A to mobile phase B is 0%:100%→5%:95%, 50-60 min, the volume ratio of mobile phase A to mobile phase B is 5%:95% → 8%:92%.

9. The method for testing the content of the longan pulp pharmaceutical preparation according to claim 8, wherein: A Shimadzu GL science Inertil ODS-3 chromatographic column with a specification of 250 mm × 4.6 mm and 5 μm is used; and / or the injection volume of the test solution is 5-20 μL; and / or the column temperature is 20-35°C and the flow rate is 0.5-1.0 mL / min.

10. The method for testing the content of the longan pulp pharmaceutical preparation according to claim 8, characterized in that: The preparation method of the test solution also satisfies any one or more of the following AE: A. The ratio of the mass of the test sample to the volume of the solvent is 0.3-0.7:10-50; the relationship between mass and volume is g / mL; B. The extraction method is reflux extraction or ultrasonic extraction; C. Extraction time is ≥10 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration; E. The solvent is water or methanol aqueous solution.

11. The method for testing the content of the longan pulp pharmaceutical preparation according to claim 10, wherein: In the preparation method of the test solution, the extraction time is 15-60 minutes.

12. The method for testing the content of the longan pulp pharmaceutical preparation according to any one of claims 8 to 10, characterized in that: The solvent used in the preparation of the uridine reference solution and the 5-hydroxymethylfurfural reference solution is selected from at least one of methanol, ethanol and water; and / or the concentration of the uridine reference solution is 0.03-50 μg / mL; and / or the concentration of the 5-hydroxymethylfurfural reference solution is 0.005-50 μg / mL.

13. A method for detecting the quality of a longan pulp pharmaceutical preparation, characterized in that: The method comprises the steps of comparing the fingerprint of the longan meat product to be tested with the control fingerprint of the longan meat pharmaceutical preparation and / or determining the content of the longan meat product to be tested by using the content testing method of the longan meat pharmaceutical preparation described in any one of claims 8 to 12; the fingerprint of the longan meat product to be tested is constructed using the longan meat product to be tested according to the construction method described in any one of claims 1 to 7, and the control fingerprint of the longan meat pharmaceutical preparation is selected from any one of the following (1) to (2): (1) A fingerprint of a pharmaceutical preparation of longan pulp obtained by using a single batch or multiple batches of pharmaceutical preparation of longan pulp according to the construction method described in any one of claims 1 to 7; (2) The fingerprints of pharmaceutical preparations of multiple batches of longan pulp obtained by the construction method described in any one of claims 1 to 7 are used to prepare a control fingerprint by using the average value or median method.

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