A method for detecting and identifying soapberry thorns and their counterfeits

Abstract

The present invention provides a method for detecting and identifying Gleditsia chinensis and its counterfeits, wherein a method for detecting Gleditsia chinensis comprises detecting with high-performance liquid chromatography, wherein the chromatographic conditions of the high-performance liquid chromatography are as follows: chromatographic column: Waters CORTECS T3C18, column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.6 μm; acetonitrile as mobile phase A, 0.1% trifluoroacetic acid aqueous solution as mobile phase B, elution according to a specific gradient elution program. The method of the present invention effectively overcomes the problems of the detection methods disclosed in the prior art, such as a small number of characteristic peaks, an unstable baseline, and very poor separation; thereby providing chromatographic conditions with a larger number of characteristic peaks, a more stable baseline, and better separation, effectively providing the prerequisites for accurately identifying Gleditsia chinensis and its counterfeits.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine detection, and in particular to a method for detecting and identifying Gleditsia sylvestris thorns and their mixed and counterfeit products. Background Art

[0002] The fingerprint or characteristic spectrum of soapberry thorn reported in the literature currently has few characteristic peaks, unstable baselines, and very poor separation of characteristic peaks, and almost no true separation is achieved.

[0003] At present, the research on distinguishing between soapberry thorn and common counterfeit products in the market, such as Japanese soapberry thorn and wild soapberry thorn, requires the establishment of separate identification methods, and all of them rely on the control of a single component. If multiple components are used for control, multiple components need to be tested. There is no way to control the soapberry thorn as a whole to distinguish between the true and the false. Even if it is identified that the sample does not contain the counterfeit component, it does not mean that the sample is soapberry thorn. Especially for pharmaceutical preparations, when the properties of the medicinal pieces are missing, the methods disclosed in the existing literature cannot completely and clearly identify that the sample is an extract of soapberry thorn, resulting in inaccurate identification of counterfeit products. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problems of the detection methods disclosed in the prior art, such as a small number of characteristic peaks, an unstable baseline and very poor separation; thereby providing a detection method for soapberry thorns that effectively provides the prerequisite for accurately judging soapberry thorns and their mixed and counterfeit products, and providing a method for identifying soapberry thorns and their mixed and counterfeit products.

[0005] In order to solve the above problems, the technical solutions provided by the present invention are as follows:

[0006] A method for detecting Gleditsia chinensis thorns, comprising detecting by high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography are:

[0007] Chromatographic column: Waters CORTECS T3 C18, column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm; acetonitrile as mobile phase A, 0.1% trifluoroacetic acid aqueous solution as mobile phase B, elution according to the following gradient elution program:

[0008]

[0009] Among the chromatographic conditions, the flow rate is 0.30-0.40 ml / min; and / or the column temperature is 33-37° C.; and / or the detection wavelength is 335 nm.

[0010] The present invention also includes the following method for preparing the test solution: taking the test substance, accurately weighing it, adding a solvent, treating it, cooling it, filtering it, and taking the filtrate to obtain it.

[0011] The present invention also includes a reference solution and / or a control solution;

[0012] The preparation method of the reference solution comprises: taking a reference solution and adding a solvent to prepare the reference solution, wherein the reference solution comprises at least one of taxifolin and quercetin;

[0013] The preparation method of the reference solution comprises: taking a reference medicinal material of Gleditsia sinensis, adding water, heating under reflux, filtering, evaporating the filtrate to dryness, adding a solvent, ultrasonically treating, cooling, shaking, filtering, and taking a subsequent filtrate to obtain the reference solution.

[0014] The solvent is at least one of methanol, ethanol and water, preferably an 80% methanol aqueous solution;

[0015] And / or, the treatment method of the test solution is ultrasonic treatment or reflux treatment; preferably, the ultrasonic treatment time is 15-45 minutes; and / or, the ultrasonic power is 250W and the frequency is 40kHz;

[0016] And / or, when the test sample is a Gleditsia sylvestris formula granule, the amount of solvent added to the test sample solution is 50 ml / g or more relative to the test sample, preferably 100-200 ml / g, more preferably 100 ml / g.

[0017] The characteristic spectrum of the test sample obtained by the high performance liquid chromatography method includes at least peak 2, peak 3, peak 5, peak 7 and peak 9; among them, peak 2 is scopoletin, peak 3 is cryptochlorogenic acid, peak 5 is taxifolin, peak 7 is orientin, and peak 9 is vitexin; taking peak 5 as the S peak, the relative retention time of the remaining characteristic peaks should be within ±10% of the specified value; the specified values ​​of the relative retention times of peak 2, peak 3, peak 7 and peak 9 are 0.45, 0.50, 1.12 and 1.37, respectively.

[0018] The characteristic spectrum of the test sample also includes neochlorogenic acid of peak 1, scopoletin of peak 4, isovitexin of peak 10, quercetin of peak 12, and scopolamine of peak 13; among which, the specified values ​​of the relative retention times of peak 1, peak 4, peak 10, peak 12 and peak 13 relative to the S peak are 0.22, 0.81, 1.52 and 2.67, respectively.

[0019] The characteristic spectrum of the test sample also includes peak 6 with specified relative retention time values ​​of 1.04, peak 8 with 1.15, and peak 11 with 1.78, respectively.

[0020] A method for identifying Gleditsia chinensis and its mixed and counterfeit products, comprising using the above-mentioned detection method to obtain characteristic patterns of Gleditsia chinensis and its mixed and counterfeit products;

[0021] If the characteristic peaks corresponding to orientin and vitexin are present, and the peak area ratio of vitexin in peak 9 to taxifolin in peak 5 is higher than 0.5, and the peak area ratio of scopoletin in peak 2 to cryptochlorogenic acid in peak 3 is higher than 0.6, then it is identified as soapberry thorn, and the rest are mixed and counterfeit products.

[0022] If there are no characteristic peaks corresponding to orientin and vitexin, and a characteristic peak appears at 34-42 minutes, it is a counterfeit product, namely, wild sapodilla thorn.

[0023] If the characteristic peaks corresponding to orientin and vitexin are present, the peak area ratio of vitexin in peak 9 to taxifolin in peak 5 is less than 0.5, and the peak area ratio of scopoletin in peak 2 to cryptochlorogenic acid in peak 3 is less than 0.6, then it is a mixed counterfeit Japanese soapberry thorn.

[0024] The technical solution of the present invention has the following advantages:

[0025] 1. The present invention provides a method for detecting the thorns of the soapberry, which can effectively detect more characteristic peaks that can be used to distinguish the thorns of the soapberry and its mixed and counterfeit products through the optimization of chromatographic conditions, especially the mutual cooperation of the gradient elution program and the mobile phase, thereby effectively providing the prerequisite for accurately judging the thorns of the soapberry and its mixed and counterfeit products.

[0026] 2. The present invention provides a method for identifying the thorns of the honey locust tree and its mixed and counterfeit products, which determines the difference between the thorns of the honey locust tree and its mixed and counterfeit products; specifically, if there are characteristic peaks corresponding to orientin and vitexin, and the peak area ratio of vitexin in peak 9 to taxifolin in peak 5 is higher than 0.5, and the peak area ratio of scopoletin in peak 2 to cryptochlorogenic acid in peak 3 is higher than 0.6, then it is identified as the thorns of the honey locust tree; if there are no characteristic peaks corresponding to orientin and vitexin, and the characteristic peak appears at 34-42 minutes, then it is the mixed and counterfeit wild honey locust tree; if there are characteristic peaks corresponding to orientin and vitexin, the peak area ratio of vitexin in peak 9 to taxifolin in peak 5 is less than 0.5, and the peak area ratio of scopoletin in peak 2 to cryptochlorogenic acid in peak 3 is less than 0.6, then it is the mixed and counterfeit Japanese honey locust tree; the above judgment rules can effectively achieve the distinction between the thorns of the honey locust tree and its different mixed and counterfeit products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 is the fingerprint of 18 batches of test solution in Example 1 of the present invention;

[0029] Figure 2 This is the reference characteristic spectrum of the Gleditsia chinensis in Example 1 of the present invention;

[0030] Figure 3 This is a comparison chart of different reference substances and the Gleditsia spica granules in Example 1 of the present invention;

[0031] Figure 4 is a chromatogram of a negative sample of the formula granules in Example 1 of the present invention;

[0032] Figure 5 is a chromatogram of a Waters CORTECS T3 C18 column in the present invention;

[0033] Figure 6 is a chromatogram of the WatersACQUITY UPLC HSS T3 column of the present invention;

[0034] Figure 7 is a chromatogram of the Agilent Poroshell 120SB C18 column of the present invention;

[0035] Figure 8 This is a comparison of the characteristic spectra of the wild honey locust thorn and the honey locust thorn in the present invention;

[0036] Figure 9 This is a comparison of the characteristic spectra of the Japanese honey locust thorn and the honey locust thorn in the present invention;

[0037] Figure 10 This is a chromatogram of the present invention when the mobile phase is acetonitrile-0.1% phosphoric acid;

[0038] Figure 11 This is a chromatogram of the present invention when the mobile phase is acetonitrile-0.1% trifluoroacetic acid;

[0039] Figure 12 This is a chromatogram of the present invention when the mobile phase is acetonitrile-0.1% formic acid;

[0040] Figure 13 This is a chromatogram of the present invention when the mobile phase is acetonitrile-0.1% acetic acid;

[0041] Figure 14 is a chromatogram of the gradient elution procedure 1 of the present invention;

[0042] Figure 15 is a chromatogram of gradient elution procedure 2 of the present invention;

[0043] Figure 16 It is the chromatogram of gradient elution procedure 3 in the present invention. DETAILED DESCRIPTION

[0044] 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.

[0045] 1. Instruments and equipment:

[0046] Chromatograph 1: Waters H-CLASS UPLC chromatography system, including quaternary solvent manager (ACQ-QSM), automatic sample injector (ACQ-FTN), original imported chromatography column oven (ACQ-CM), diode array UV detector (ACQ-PDA), and Empower chromatography management system;

[0047] Chromatograph 2: Thermo Vanquish Flex UPLC chromatography system, including a quaternary solvent manager (VanquishQuaternary Pump F VF-P20-A), an autosampler (Vanquish Split Sampler FT VF-A10-A-02), an imported column oven (Vanquish Column Compartment H VH-C10-A-02), and a DAD detector (Vanquish VF-D40-A).

[0048] XS204, XS205, XSE205 ten-thousandth balances (Mettler, Switzerland), ME36S millionth balance (Mettler, Switzerland), ultrasonic analyzer (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), water bath.

[0049] 2. Reagents

[0050] Acetonitrile (Fisher Chemical, chromatographic grade) and water were ultrapurified water, and other reagents such as trifluoroacetic acid and methanol were of analytical grade.

[0051] 3. Drug testing

[0052] Gleditsia sinensis control medicinal material (batch number: 121210-201504, purchased from China Food and Drug Inspection Institute);

[0053] Quercetin reference substance (batch number: 100081-201610, purchased from China Food and Drug Administration, purity 99.1%);

[0054] Taxifolin reference substance (batch number: 111816-201102, purchased from China Food and Drug Administration, purity 98.9%);

[0055] Orientin reference substance (batch number: 111777-202003, purchased from China Food and Drug Administration, purity 98.0%);

[0056] Vitexin reference substance (batch number: 111687-202105, purchased from China Food and Drug Administration, purity 99.1%);

[0057] Isovitexin reference substance (batch number: 250215-202110, purchased from Shanghai Hongyong Biotechnology Co., Ltd., purity ≥98%);

[0058] Cryptochlorogenic acid reference substance (lot number: G153101, purchased from Aladdin);

[0059] New chlorogenic acid reference substance (batch number: 19011731, purchased from TAUTO, purity 98.0%);

[0060] Scopoletin reference substance (batch number: 110768-202105, purchased from China Food and Drug Inspection Institute);

[0061] Scopoletin reference substance (batch number: 040006-202209, purchased from Shanghai Hongyong Biotechnology Co., Ltd., purity ≥98%);

[0062] Babiquinone reference substance (batch number: 001743-202205, purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd., purity ≥98%);

[0063] Gleditsia sinensis formula granules (batch numbers: 1901001Y, 1901002Y, 1901003Y).

[0064] Example 1

[0065] A method for detecting the thorns of the honey locust tree can effectively obtain characteristic spectra of the honey locust tree thorns and their mixed and counterfeit products. The specific acquisition process is as follows:

[0066] 1. Feature map acquisition

[0067] Based on the Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules and the technical requirements for the research and formulation of traditional Chinese medicine quality standards in the Chinese Pharmacopoeia, and referring to the established chromatographic conditions for the determination of the characteristic spectrum of the standard decoction of Gleditsia sinensis slices, the chromatographic conditions and system adaptability of the characteristic spectrum determination method of Gleditsia sinensis formula granules were studied and verified, and the methodology was investigated, and a characteristic spectrum determination method for Gleditsia sinensis formula granules was established.

[0068] The chromatographic conditions and system suitability experiments were as follows: octadecylsilane bonded silica gel was used as the packing material (Waters CORTECS T3 C18, column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A, and 0.1% trifluoroacetic acid solution was used as mobile phase B, with gradient elution as specified in Table 1 below; the flow rate was 0.35 ml / min; the column temperature was 35°C; and the detection wavelength was 335 nm.

[0069] Table 1 Gradient elution table

[0070]

[0071] Preparation of reference solution: Take about 2 g of Gleditsia sinensis reference medicinal material, place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of 80% methanol to the residue, stopper it, and ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the reference solution of the control medicinal material.

[0072] Preparation of reference solution: Take appropriate amount of Taxifolin reference substance and Quercetin reference substance, weigh accurately, add methanol to make mixed solutions containing 40 μg Taxifolin and 30 μg Quercetin per 1 ml, respectively, to obtain reference solution.

[0073] Preparation of test solution: Weigh about 0.2 g of Gleditsia sinensis granules, accurately weigh, place in a stoppered conical flask, accurately add 20 mL of 80% methanol, stopper, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate.

[0074] Determination method: Accurately aspirate 1 μl of the test solution, reference solution and reference solution respectively, inject them into the liquid chromatograph, and determine them.

[0075] The above test samples included 15 batches of standard decoction of Gleditsia sinensis slices (lyophilized powder) and 3 batches of Gleditsia sinensis formula granules. Fingerprints were obtained through 18 batches of test sample solutions, such as Figure 1 As shown, S1-S15 are standard decoctions of Gleditsia chinensis slices, and S16-S18 are formula granules of Gleditsia chinensis. The fingerprint detection results are analyzed by using the fingerprint similarity evaluation software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee, and the "multi-point correction, MARK peak matching" mode is used for fitting and generating a reference characteristic spectrum. The reference characteristic spectrum of Gleditsia chinensis is obtained, as shown in FIG. Figure 2 shown.

[0076] 2. Identification and identification of characteristic peaks

[0077] Figure 2 The HPLC characteristic spectrum of the granules of the Chinese honey locust tree obtained in the experiment has a total of 13 chromatographic peaks. HPLC and LC / MS / MS were used to identify and assign the characteristic peaks. Among them, HPLC determined that peak 1 was neochlorogenic acid, peak 2 was scopoletin, peak 3 was cryptochlorogenic acid, peak 4 was scopoletin, peak 5 was taxifolin, peak 7 was orientin, peak 9 was vitexin, peak 10 was isovitexin, peak 12 was quercetin, and peak 13 was scopolamine. Figure 3 Peaks 1 to 13 were analyzed by LC / MS / MS, confirming that their molecular structures were consistent with those of reference substances: neochlorogenic acid, scopoletin, cryptochlorogenic acid, scopoletin, taxifolin, orientin, vitexin, isovitexin, quercetin, and scopolamine. The results are shown in Table 2 below.

[0078] Table 2 LC / MS / MS analysis results of Gleditsia chinensis

[0079]

[0080] In summary, the characteristic spectrum of the Gleditsia sinensis granules, identified by HPLC and LC / MS / MS, confirmed that Peak 1 was neochlorogenic acid, Peak 2 was scopoletin, Peak 3 was cryptochlorogenic acid, Peak 4 was scopoletin, Peak 5 was taxifolin, Peak 7 was orientin, Peak 9 was vitexin, Peak 10 was isovitexin, Peak 12 was quercetin, and Peak 13 was scopolamine. Therefore, the characteristic spectrum of the Gleditsia sinensis granules reflects the characteristics of the main chemical components and active ingredients of Gleditsia sinensis.

[0081] 3. Determination of characteristic chromatographic reference peak (S peak) and relative retention time

[0082] According to the peak identification and peak selection results of the characteristic peaks in the Chinese honeysuckle granules, peak 1 was determined to be neochlorogenic acid, peak 2 to scopoletin, peak 3 to cryptochlorogenic acid, peak 4 to scopoletin, peak 5 to taxifolin, peak 7 to orientin, peak 9 to vitexin, peak 10 to isovitexin, peak 12 to quercetin, and peak 13 to serpentine. Among them, taxifolin and quercetin were the index components for content determination, and the retention time of taxifolin was relatively moderate. Therefore, taxifolin was used as the reference peak to better evaluate the relative retention time of each characteristic peak.

[0083] The relative retention times of the 13 characteristic peaks are all within ±10% of the specified values, and the specified values ​​are: 0.22 (peak 1), 0.45 (peak 2), 0.50 (peak 3), 0.81 (peak 4), 1.04 (peak 6), 1.12 (peak 7), 1.15 (peak 8), 1.37 (peak 9), 1.52 (peak 10), 1.78 (peak 11), and 2.67 (peak 13).

[0084] 4. Chromatographic conditions and system suitability

[0085] Prepare the test solution according to the solution method according to the test solution preparation method, and examine whether the negative sample of the Chinese honey locust granules will cause interference. Perform HPLC analysis on the negative sample of the Chinese honey locust granules, the mixed reference substance, the Chinese honey locust granules and the Chinese honey locust reference medicinal materials according to the above chromatographic conditions, record the chromatogram of the negative sample, and confirm the overall chromatogram results such as the peak area, separation degree and other characteristics of the mixed reference substance and the reference medicinal materials. Figure 4 As shown in Table 3-Table 4. Among them, the negative sample of the formula granule is the negative granule compressed from the auxiliary material maltodextrin of the formula granule.

[0086] Table 3 Overall chromatogram results of mixed reference substances and Gleditsia sinensis reference medicinal materials

[0087]

[0088]

[0089] Table 4 Overall chromatogram results of Gleditsia sinensis formula granules

[0090]

[0091] Depend on Figure 4 The blank control sample showed no interference with the characteristic spectrum, indicating that the chromatographic method exhibited good system adaptability and specificity, making it suitable for use as a method for detecting the characteristic spectrum of the Gleditsia sinensis granules. Tables 3 and 4 indicate that the Gleditsia sinensis granules and the Gleditsia sinensis control herb exhibit numerous characteristic peaks with good resolution.

[0092] Example 2

[0093] A method for identifying Gleditsia sylvestris and its mixed and counterfeit products is as follows:

[0094] According to literature research and field investigations, there are many counterfeit products of soapberry thorn, mainly including Japanese soapberry thorn and wild soapberry thorn.

[0095] Wild honey locust thorns are spines found on the branches of the legume plant Gleditsia heterophylla Bunge. Because the pods are short, they are also known as short-horned honey locusts. The main spine is small, about 0.6-6 cm long, 0.1-0.4 cm in diameter at the base, and pointed at the end. It often has a pair of short branches, though a few are unbranched, each 0.2-0.8 cm long and about 0.1 cm in diameter. The entire spine is reddish-brown or tan in color. It is light, hard, and easily broken, with a faint odor and a mild taste.

[0096] Japanese soapberry, also known as mountain soapberry, is a thorn of the leguminous plant Glrditsia japonica Miq. The main spine is 3.5–17 cm long, 0.2–0.5 cm thick at the base, tapering upward to a sharp point. Branching spines, mostly located below the main spine, are 0.4–6 cm long and 0.3–0.5 cm in diameter. The entire spine is reddish-brown or purple-brown with a slight sheen. It is light, hard, and easily broken. The wood in cross section is light yellow-brown, and the pith is large, loose, and light reddish-brown. It has a faint odor and a bitter taste.

[0097] Therefore, the characteristic spectrum method of Gleditsia sinensis formula particles was used to test the wild Gleditsia sinensis and Japanese Gleditsia sinensis samples. The test results are as follows: Figure 8 and Figure 9 shown.

[0098] pass Figure 8 It can be seen that the characteristic spectrum of wild soapberry thorn is quite different from that of soapberry thorn. Compared with the 13 characteristic peaks of soapberry thorn, wild soapberry thorn lacks characteristic peaks 7 and 9, and at 34-42 minutes, wild soapberry thorn shows obvious characteristic peaks, indicating that the two have obvious differences in chemical composition.

[0099] pass Figure 9 It can be seen that the characteristic spectra displayed by the Japanese honey locust thorn and the honey locust thorn are basically the same. However, the peak area ratios of Peak 2 (scopoletin) / Peak 5 (taxifolin), Peak 9 (vitexin) / Peak 5 (taxifolin), Peak 10 (isovitexin) / Peak 5 (taxifolin), and Peak 2 (scopoletin) / Peak 3 (cryptochlorogenic acid) in the honey locust thorn sample are somewhat different from those of the Japanese honey locust thorn and the wild honey locust thorn. Because the peak area ratios of Peak 2 (scopoletin) / Peak 5 (taxifolin) and Peak 10 (isovitexin) / Peak 5 (taxifolin) in the honey locust thorn sample are not obvious for distinguishing from mixed products, the peak area ratios of Peak 9 (vitexin) / Peak 5 (taxifolin) and Peak 2 (scopoletin) / Peak 3 (cryptochlorogenic acid) are used for differentiation.

[0100] The same chromatographic conditions as in Example 1 were used to analyze the peak areas and relative peak areas of multiple batches of samples. The peak area ratio of Peak 9 (vitexin) to Peak 5 (taxifolin) in the Spina Gleditsiae samples was 0.57-2.05, and the peak area ratio of Peak 9 (vitexin) to Peak 5 (taxifolin) in the Spina Japonicae was 0.34-0.47, indicating a significant difference in the peak area ratios. At the same time, the peak area ratio of Peak 2 to Peak 3 in the Spina Gleditsiae samples ranged from 0.71 to 4.35, and the peak area ratio of Peak 2 to Peak 3 in the Spina Japonicae was 0.39-0.56, indicating a significant difference in the peak area ratios. Therefore, the relative peak area ratio of Peak 2 to Peak 3 and the peak area ratio of Peak 9 to Peak 5 can be used to distinguish Spina Gleditsiae from Spina Japonicae.

[0101] In summary, Peak 2, Peak 3, Peak 5, Peak 7 and Peak 9 can be used to effectively distinguish between common counterfeit products of Sophora japonica, Sophora japonica and Sophora japonica. Specifically, according to the lower limit of the measured range of multiple batches of Sophora japonica samples multiplied by the coefficient 0.9, it is stipulated that the relative peak area of ​​Peak 9 and Peak 5 in the Sophora japonica formula granule sample shall not be less than 0.5 (0.57×0.9=0.51, rounded to 0.5); and the relative peak area of ​​Peak 2 and Peak 3 shall not be less than 0.6 (0.71×0.9=0.64, rounded to 0.6).

[0102] Example 3

[0103] The precision and robustness of the chromatographic conditions in Example 1 were investigated.

[0104] 1. Precision

[0105] 1.1 Instrument precision test

[0106] The same sample solution of the Gleditsia sinensis formula granules was taken and injected 6 times according to the chromatographic conditions of Example 1. The relative retention time and relative peak area of ​​each common peak were measured, as shown in Tables 5 and 6 below, where the peak area is represented by M and the relative peak area is represented by XM.

[0107] Table 5 Instrument precision relative retention time test results

[0108]

[0109] Table 6 Instrument precision relative peak area test results

[0110]

[0111] The results in Tables 5 and 6 show that the RSDs of the relative retention times of the characteristic peaks and the reference peaks are all less than 2%, indicating that the instrument has good precision.

[0112] 1.2 Method repeatability test

[0113] The same portion of the Gleditsia sinensis formula granules was taken and 6 portions were prepared repeatedly according to the test sample preparation method in Example 1. The relative retention time and relative peak area of ​​each common peak were measured according to the chromatographic conditions. The test results are shown in Tables 7 and 8 below, where the peak area is represented by M and the relative peak area is represented by XM.

[0114] Table 7 Method repeatability relative retention time test results

[0115]

[0116]

[0117] Table 8 Method repeatability relative peak area test results

[0118]

[0119] The above results show that the RSD of the relative retention time of each characteristic peak and the reference peak is less than 2%, indicating that the method has good repeatability.

[0120] 1.3 Intermediate precision (different operators A, B, C)

[0121] Three inspectors took the same portion of Gleditsia sylvestris formula granules at different times, prepared samples according to the test sample preparation method in Example 1, and used the same equipment to measure the relative retention time and relative peak area of ​​each common peak. The test results are shown in Tables 9 and 10, where the peak area is represented by M and the relative peak area is represented by XM.

[0122] Table 9 Intermediate precision relative retention time test results (different operators)

[0123]

[0124]

[0125] Table 10 Intermediate precision relative peak area test results (different operators)

[0126]

[0127] The above results show that the relative average deviations of the relative retention times of the characteristic peaks and the reference peaks are all less than 2%, indicating that the intermediate precision of the method is good.

[0128] 2. Durability

[0129] 2.1 Stability investigation

[0130] The same sample solution of the Gleditsia spica formula granules was taken and injected at 0, 4, 8, 12, 18, and 24 hours according to the chromatographic conditions in Example 1. The relative retention time and relative peak area of ​​each common peak were measured. The test results are shown in Tables 11 and 12, where the peak area is represented by M and the relative peak area is represented by XM.

[0131] Table 11 Stability relative retention time test results

[0132]

[0133]

[0134] Table 12 Stability relative peak area test results

[0135]

[0136] The above results show that the deviation of the relative retention time of each chromatographic peak and the reference peak is less than 2%, indicating that the test solution is stable within 24 hours and meets the determination requirements.

[0137] 2.2 Investigation of different column temperatures

[0138] The same sample solution of the Gleditsia spica formula granules was taken, and the column temperatures were set to 33°C, 35°C, and 37°C according to the chromatographic method in Example 1. The effects of different column temperatures on the relative retention time and relative peak area of ​​each characteristic peak were investigated. The test results are shown in Tables 13 and 14, where the peak area is represented by M and the relative peak area is represented by XM.

[0139] Table 13 Comparison of relative retention time results at different column temperatures

[0140]

[0141] Table 14 Comparison of relative peak area results at different column temperatures

[0142]

[0143]

[0144] The results showed that when the column temperature changed, the deviation of the relative retention time of each characteristic chromatographic peak was within 2%. The column temperature would affect the peak shape and relative peak area of ​​each characteristic chromatographic peak, but the deviation of the relative peak area of ​​the characteristic peaks of Peak 2, Peak 3, Peak 5 and Peak 9 was within 10%. Therefore, this temperature range is suitable for the distinction between Gleditsia sylvestris and its mixed products. Based on the separation of other characteristic peaks, the preferred column temperature is 35℃.

[0145] 2.3 Investigation of different flow rates

[0146] The same sample solution of the Gleditsia spica formula granules was taken and measured according to the determination method of Example 1 at flow rates of 0.30 ml / min, 0.35 ml / min, and 0.40 ml / min, respectively, to investigate the effect of slight changes in the flow rate on the relative retention time and relative peak area of ​​each characteristic peak. 0.30 ml / min was numbered 1, 0.35 ml / min was numbered 2, and 0.40 ml / min was numbered 3. The test results are shown in Tables 15 and 16, where the peak area is represented by M and the relative peak area is represented by XM.

[0147] Table 15 Comparison of relative retention time results at different flow rates

[0148]

[0149] Table 16 Comparison of relative peak area results at different flow rates

[0150]

[0151]

[0152] These results indicate that slight changes in flow rate can lead to significant variations in the retention times of characteristic chromatographic peaks, and their relative peak areas also change. However, the deviations in the relative retention times of Peaks 2, 3, 5, 7, and 9 are all within 2%, and the deviations in the relative peak areas of Peaks 2, 3, 5, and 9 are all within 10%. Therefore, this flow rate range is suitable for distinguishing Gleditsia sinensis from its adulterants. At 0.35 ml / min, the resolution of Peaks 7 and 8 is better, making a fixed flow rate (0.35 ml / min) the preferred method for determination.

[0153] 2.4. Investigation of different instruments

[0154] The same sample solution of the Gleditsia spica formula granules was taken and analyzed using different instruments (waters, Thermo) according to the determination method in Example 1 to investigate the effects of different instruments on the relative retention time and relative peak area of ​​each characteristic peak. Waters was numbered 1 and Thermo was numbered 2. The test results are shown in Tables 17 and 18, where the peak area is represented by M and the relative peak area is represented by XM.

[0155] Table 17 Comparison of relative retention time results of different instruments

[0156]

[0157] Table 18 Comparison of relative peak area results of different instruments

[0158]

[0159] The above results show that when using instruments from different manufacturers, there is a certain impact on the relative retention time of each characteristic chromatographic peak, but it is within the range of ±10% of the specified value. Therefore, different instruments are applicable.

[0160] 2.5. Investigation of different chromatographic columns of the same batch

[0161] The same batch of test solution was taken, and the determination method of Example 1 was used to set up two chromatographic columns of different batches, Waters CORTECS T3 C18, namely, batch number SN: 01213123715316 (number 1) and batch number SN: 01213133416821 (number 2). The relative retention time and relative peak area of ​​each characteristic peak and peak 5 (S) were investigated when the batch number of the chromatographic column was changed. The results are shown in Tables 19 and 20 below, where the peak area is represented by M and the relative peak area is represented by XM.

[0162] Table 19 Comparison of relative retention time results of different chromatographic columns

[0163]

[0164] Table 20 Comparison of relative peak area results of different chromatographic columns

[0165]

[0166] The above results show that the average deviation of the relative retention times is less than 2%. The results show that the retention times and relative peak areas of the characteristic chromatographic peaks vary little with column batches. Therefore, the recommended fixed column for this method is: Waters CORTECS T3 C18 (2.1mm×100mm, 1.6μm).

[0167] 2.6. Sample injection volume investigation

[0168] Accurately weigh 0.2 g of Gleditsia sinensis granules and place them in a stoppered conical flask. Accurately add 20 mL of 80% methanol and weigh the mixture. Ultrasonicate (power 250 W, frequency 40 kHz) for 30 minutes. Allow to cool, then weigh again. Make up the loss with 80% methanol, shake well, and filter to obtain the test solution. Accurately pipette 0.5 μL, 1 μL, 1.5 μL, and 2 μL of the above solution into an ultrahigh performance liquid chromatograph and record the chromatogram.

[0169] The recorded chromatograms show that the peak area increases with increasing injection volume. Sample concentration overload occurs at 1.5 μL and 2 μL. Compared to 0.5 μL, a 1.0 μL injection volume produces better chromatographic peak response and system suitability parameters. Therefore, a 1.0 μL injection volume is preferred for the characteristic chromatographic method of Gleditsia sinensis granules.

[0170] Example 4

[0171] 1. Detection results of different gradient elution programs

[0172] The gradient elution programs in Tables 23 to 25 below were used for detection, and the test results were as follows: Figure 14-16 shown.

[0173] Table 23 Gradient elution program 1

[0174]

[0175] Table 24 Gradient elution program 2

[0176]

[0177] Table 25 Gradient elution program 3

[0178]

[0179] The above results show that when elution gradients 1 and 2 are used, the separation of characteristic peaks is poor, the baseline is unstable, and the detection time is long. However, when elution gradient 3 is used, the separation of characteristic peaks in the obtained spectrum is better, the baseline is stable, and the detection time is short.

[0180] 2. Test results of different chromatographic columns

[0181] The same batch of test solution was taken and the determination method in Example 1 was used to set up chromatographic columns Waters CORTECST3C18 (2.1mm×100mm, 1.6μm), Waters ACQUITY UPLC HSS T3 (2.1mm×100mm, 1.8μm) and Agilent Poroshell 120SB C18 (2.1*100mm, 1.9μm). The relative retention time and relative peak area of ​​each characteristic peak and peak 5 (S) were investigated on different chromatographic columns. The test results are shown in FIG. Figure 5-Figure 7 As shown. Figure 5-Figure 7 The results show that Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) and Agilent Poroshell 120SB C18 (2.1*100 mm, 1.9 μm) cannot reproduce the modified method; therefore, the fixed chromatographic column of the present invention is: Waters CORTECS T3 C18 (2.1 mm × 100 mm, 1.6 μm).

[0182] 3. Investigation of different mobile phases

[0183] Take the test solution of the Gleditsia spp. formula granules and inject it into the liquid chromatograph. According to the chromatographic conditions in Example 1, compare the chromatograms of four different mobile phase systems: acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% trifluoroacetic acid, acetonitrile-0.1% formic acid and acetonitrile-0.1% acetic acid. The test results are as follows: Figure 10-13 shown.

[0184] pass Figure 10-13 It can be seen that when using acetonitrile-0.1% trifluoroacetic acid mobile phase system, the separation between chromatographic peaks 1, 2, 3, 6, 12 and 13 is significantly better than that of phosphoric acid, acetic acid and formic acid at the same concentration, and the peak information content of the characteristic spectrum under trifluoroacetic acid conditions is relatively large. Therefore, acetonitrile-0.1% trifluoroacetic acid solution system is selected as the mobile phase system for the characteristic spectrum of Gleditsia sinensis formula granules.

[0185] 4. Investigation of different acid concentration ratios

[0186] The same sample solution of the Gleditsia sinensis formula granules was taken and measured according to the determination method in Example 1 using 0.05% trifluoroacetic acid (numbered 1), 0.1% trifluoroacetic acid (numbered 2), and 0.2% trifluoroacetic acid (numbered 3). The effects of slight changes in the acid concentration ratio on the relative retention time and relative peak area of ​​each characteristic peak were investigated. The results are shown in Tables 26 and 27.

[0187] Table 26 Comparison of relative retention time results for different acid concentration ratios

[0188]

[0189] Table 27 Comparison of relative peak area results of different acid concentration ratios

[0190]

[0191] These results demonstrate that even small changes in the acid concentration ratio have a certain impact on chromatographic peak shape, relative peak area, and resolution. The relative retention time deviations for the characteristic peaks of Peaks 2, 3, 5, 7, and 9 are all within 2%, while the relative peak area deviations for the characteristic peaks of Peaks 2, 3, and 9 are all above 10%. Therefore, based on the results of investigations at different acid concentration ratios and relative peak areas, this method utilizes 0.1% trifluoroacetic acid for determination.

[0192] Example 5

[0193] 1. Investigation of extraction solvent

[0194] Compare the addition of different extraction solvents to the test solution and determine the appropriate extraction solvent based on parameters such as peak information content, peak area, and peak separation.

[0195] Take 0.2g of the Gleditsia spp. granules, accurately weigh them, place them in a stoppered conical flask, accurately add 20mL each of water, ethanol, methanol, 50% methanol, and 80% methanol, stopper them tightly, weigh them, and ultrasonically treat them (power 250W, frequency 40kHz), heat them under reflux for 30 minutes, cool them, weigh them again, make up the lost weight with the added solvent, shake them well, and filter them to obtain the test solution. Accurately pipette 1μL of the above solution into an ultra-high performance liquid chromatograph, and record the test results as shown in Table 28 below.

[0196] Table 28

[0197]

[0198]

[0199] A comparative study of different extraction solvents showed that the chromatograms using methanol and water as solvents were better than the chromatogram using ethanol as solvent. When 80% methanol was used as the mobile phase system, the total area of ​​the chromatographic peaks was higher than that of other solvent systems. Based on the separation and symmetry of the spectral peaks, 80% methanol was selected as the solvent system for the characteristic spectrum method of the Gleditsia sinensis formula particles, which was used for the investigation and screening of subsequent methods.

[0200] 2. Investigation of treatment methods

[0201] Compare the two treatment methods of ultrasonic treatment and heating reflux, and determine the appropriate extraction method based on parameters such as peak information volume, peak area, and peak separation.

[0202] Specifically, 0.2 g of the granules of the honey locust tree were accurately weighed and placed in a stoppered conical flask. 20 mL of 80% methanol was accurately added, the flask was sealed, and the weight was determined. Ultrasonic treatment (power 250 W, frequency 40 kHz) and reflux were performed for 30 minutes, respectively. The mixture was cooled and weighed again. The weight loss was supplemented with 80% methanol, shaken well, and filtered to obtain the test solution. 1 μL of the above solution was accurately aspirated and injected into an ultra-high performance liquid chromatograph. The test results are shown in Table 29.

[0203] Table 29

[0204]

[0205]

[0206] Ultrasonic and reflux extraction methods are used. When ultrasonic treatment is used, the peak area of ​​the sample peak is higher than that of reflux treatment, and the sample extraction is more complete. Ultrasonic treatment (power 250W, frequency 40KHz) is preferably used.

[0207] 3. Investigation of extraction time

[0208] Take 0.2g of the granules of the Chinese honey locust tree, accurately weigh them, place them in a stoppered conical flask, accurately add 20mL of 80% methanol, weigh them, and sonicate them (power 250W, frequency 40kHz) for 15, 30, and 45 minutes, respectively. Let them cool, weigh them again, make up the lost weight with 80% methanol, shake well, and filter to obtain the test solution. Accurately pipette 1μL of the above solution and inject it into an ultrahigh performance liquid chromatography instrument. The test results show that there is no significant difference in the response value of the characteristic peak and the system suitability parameters under different sonication conditions. Taking all factors into consideration, the sonication time is selected to be 30 minutes.

[0209] 4. Investigation of different sampling amounts

[0210] Accurately weigh 0.1, 0.2, and 0.4 g of each of the Gleditsia sinensis granules. Place in a stoppered conical flask. Accurately add 20 mL of 80% methanol and weigh the sample. Ultrasonicate (power 250 W, frequency 40 kHz) for 30 minutes. Allow to cool, weigh again, and make up the lost weight with 80% methanol. Shake well and filter to obtain the test solution. Accurately pipette 1 μL of this solution and inject it into an ultrahigh performance liquid chromatograph. Sample sizes of 0.1 g, 0.2 g, and 0.4 g were used. As the sample size increased, the total peak area of ​​the characteristic peaks increased proportionally.

[0211] 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 detecting Gleditsia chinensis thorns, characterized in that: The method includes detecting by ultra-high performance liquid chromatography, wherein the chromatographic conditions of the ultra-high performance liquid chromatography are: Chromatographic column: Waters CORTECS T3 C18, column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm; acetonitrile as mobile phase A, 0.1% trifluoroacetic acid aqueous solution as mobile phase B, elution according to the following gradient elution program: The detection wavelength is 335 nm; The preparation method of the test solution is as follows: taking the substance to be tested, accurately weighing it, adding a solvent, treating it, cooling it, filtering it, and taking the filtrate to obtain it; The solvent is at least one of methanol, ethanol and water; The reference substances include quercetin reference substance, taxifolin reference substance, orientin reference substance, vitexin reference substance, isovitexin reference substance, cryptochlorogenic acid reference substance, neochlorogenic acid reference substance, scopoletin reference substance, scopoletin reference substance, and scopolamine reference substance.

2. The detection method according to claim 1, wherein Among the chromatographic conditions, the flow rate is 0.30-0.40 ml / min; and / or the column temperature is 33-37°C.

3. The detection method according to claim 1 or 2, characterized in that Also included are reference solutions and / or control solutions; The preparation method of the reference substance solution comprises: taking a reference substance and adding a solvent to prepare the solution; The preparation method of the reference solution comprises: taking a reference medicinal material of Gleditsia sinensis, adding water, heating under reflux, filtering, evaporating the filtrate to dryness, adding a solvent, ultrasonically treating, cooling, shaking, filtering, and taking a subsequent filtrate to obtain the reference solution.

4. The detection method according to claim 1, wherein The solvent is 80% methanol aqueous solution; And / or, the test solution is treated by ultrasonic treatment or reflux treatment; And / or, when the test sample is a granule of Gleditsia sinensis, the amount of solvent added to the test sample solution is 50 ml / g or more relative to the test sample.

5. The detection method according to claim 4, characterized in that The ultrasonic treatment time is 15-45 minutes; and / or the ultrasonic power is 250W and the frequency is 40kHz; And / or, when the test sample is a Gleditsia sylvestris formula granule, the amount of solvent added to the test sample solution is 100-200 ml / g relative to the test sample.

6. The detection method according to claim 1 or 2, characterized in that The characteristic spectrum of the test sample obtained by the ultra-high performance liquid chromatography method includes at least peak 2, peak 3, peak 5, peak 7 and peak 9; among them, peak 2 is scopoletin, peak 3 is cryptochlorogenic acid, peak 5 is taxifolin, peak 7 is orientin, and peak 9 is vitexin; taking peak 5 as the S peak, the relative retention time of the remaining characteristic peaks should be within ±10% of the specified value; the specified values ​​of the relative retention times of peak 2, peak 3, peak 7 and peak 9 are 0.45, 0.50, 1.12 and 1.37, respectively.

7. The detection method according to claim 6, characterized in that The characteristic spectrum of the test sample also includes neochlorogenic acid of peak 1, scopoletin of peak 4, isovitexin of peak 10, quercetin of peak 12, and scopolamine of peak 13; among which, the specified values ​​of the relative retention times of peak 1, peak 4, peak 10, peak 12 and peak 13 relative to the S peak are 0.22, 0.81, 1.52 and 2.67, respectively.

8. The detection method according to claim 6, characterized in that The characteristic spectrum of the test sample also includes peak 6 with specified relative retention time values ​​of 1.04, peak 8 with 1.15, and peak 11 with 1.78, respectively.

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