Specific chromatogram identification method for sword beans and counterfeit sword beans

Abstract

The invention provides a specific chromatogram identification method for sword beans and counterfeit products thereof. The specific chromatogram identification method comprises the following steps: a, taking a to-be-detected sample for pretreatment; b, determining the to-be-detected liquid by adopting a high performance liquid chromatography to obtain HPLC characteristic chromatograms of the sword beans and the preparation thereof, c, comparing characteristic peaks, and if the relative retention time of the to-be-detected sample corresponds to the characteristic peaks, determining that the to-be-detected sample is a certified sword bean product; if the relative retention time does not correspond to the characteristic peak, the sword beans are counterfeit sword beans. The method for identifying the specific chromatograms of the sword beans and counterfeit mucuna sempervirens seeds of the sword beans is established by adopting ultra-high performance liquid chromatography for the first time, and a contrast specific chromatogram of the specific chromatograms of sword bean medicinal materials is established. The method has the characteristics of high operability, convenience in analysis and stability. According to the invention, the ultra-high performance liquid chromatography fingerprint technology is applied to control the identification of sword beans and counterfeit products thereof, so that the quality of the raw material medicine is effectively controlled.

Description

Technical Field

[0001] The invention relates to a characteristic spectrum identification method for sword bean and its counterfeit products, and belongs to the field of traditional Chinese medicine identification. Background Art

[0002] The use of sword beans was documented as early as the Ming Dynasty's "Compendium of Materia Medica." Shizhen's "Compendium of Materia Medica" states: "The sword bean is named after its pod shape... When it matures, the seeds, which are thumb-sized and light red, are collected." It is sweet and warm in nature and enters the stomach and kidney meridians. It has the effects of warming the middle, lowering qi, and relieving hiccups, and is used for hiccups and vomiting caused by cold. Sword beans are the dried, mature seeds of the legume plant Canavalia gladiata (Jacq.) DC. Currently, the seeds of the same family, Mucuna sempervirens Hemsl., are used in place of sword beans in the market. Literature: Wang Shumei, Ding Qing. Identification of Sword Bean Counterfeit and Hedera oleifera Seeds[J]. Chinese Modern Applied Pharmacy, 2001, (03): 195-196. DOI: 10.13748 / j.cnki.issn1007-7693.2001.03.011. Zhang Shuren. Identification of Sword Bean and Its Counterfeit Products[J]. Jilin Traditional Chinese Medicine, 1998, (04): 50. The two products were identified from the aspects of source, properties, microscopy and physicochemical properties. There were systematic defects in subjectivity, specificity and anti-interference ability. Wu Yue, Ding Qing. Identification of Sword Bean and Its Counterfeit Products by SDS-PAGE[J]. Strait Pharmacy, 2007, (07): 62-63. The two products were identified by SDS-PAGE gel electrophoresis. Among them, the SDS-PAGE method has limited distinguishing ability and sometimes cannot completely separate different medicinal materials. The spectral band characteristics are not obvious, the separation degree from adjacent spectral bands is not high, and the significance is not strong, which will affect the accuracy of identification. In addition, the protein bands of samples from different origins vary in distribution, quantity, staining degree, etc., which may be related to the type of protein, processing method, storage conditions, etc., affecting the consistency of the results. Summary of the Invention

[0003] The present invention uses UPLC technology to establish an effective method for identifying Jack Bean and its counterfeit product, Hedera helix seeds, in order to provide a reference for further improving and perfecting the quality control standards of Jack Bean raw materials and ensuring the safety of Jack Bean clinical use, and to lay a foundation for subsequent research work on the identification of Jack Bean authenticity.

[0004] The present invention provides a method for identifying sword beans and their counterfeits using characteristic patterns, which comprises the following steps:

[0005] a. Pre-treatment of samples to be tested;

[0006] b. The liquid to be tested is measured by high performance liquid chromatography to obtain HPLC characteristic spectrum of sword bean and its preparation;

[0007] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an acetonitrile solution, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the gradient elution is performed;

[0008] The gradient elution is specifically as follows:

[0009] 0-12 min, phase A: 10→14%, phase B: 90→86%;

[0010] 12-22 min, phase A: 14% → 16%, phase B: 86% → 84%;

[0011] 22-25 min, phase A: 16% → 18%, phase B: 84% → 82%;

[0012] 25-45 min, phase A: 18% → 30%, phase B: 82% → 70%;

[0013] 45-50 min, phase A: 30%, phase B: 70%;

[0014] c. Compare the characteristic peaks. The relative retention time of the characteristic peaks is:

[0015] Peak 1: 0.47, peak 2: 0.88, peak 3: 0.96, peak 5: 1.52, peak 6: 1.56, peak 7: 1.58, peak 8: 1.61, the relative retention times are within ±10% of the specified values;

[0016] If the relative retention time of the sample to be tested corresponds to the above-mentioned characteristic peak, it is a genuine sword bean; if the relative retention time does not correspond to the above-mentioned characteristic peak, it is a counterfeit sword bean.

[0017] Among them, the pretreatment method of the sample to be tested in step a is:

[0018] Take the sample to be tested, add methanol, ultrasonically treat, cool, filter, and take the filtrate.

[0019] Preferably, the methanol concentration is 80%, and the ultrasonic treatment conditions are: power 600W, frequency 40kHz; and treatment time 30 minutes.

[0020] The chromatographic column is C18 150×2.1mm 1.8μm; the column temperature is 20-35°C; the theoretical plate number calculated based on rutin should be no less than 5000; the mobile phase flow rate is 0.2-0.3mL / min; the injection volume is 1-3μL; and the detection wavelength is 340-360nm.

[0021] Preferably, the column temperature is 25° C.; the mobile phase flow rate is 0.2 mL / min; the injection volume is 2 μL; and the detection wavelength is 340 nm.

[0022] Wherein, the counterfeit sword bean is: Mucuna sempervirens Hemsl. seeds.

[0023] This invention, for the first time, uses ultra-high performance liquid chromatography (UPLC) to establish a method for identifying Jack Bean (canavalia serrata) and its counterfeit, Hedera oleifera seeds, using a characteristic spectrum. This method also establishes a comparative characteristic spectrum for the Jack Bean medicinal material. This method is characterized by strong operability, convenient analysis, and stability. The invention utilizes UPLC fingerprinting technology to control the identification of Jack Bean and its counterfeit products, effectively controlling the quality of the API. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Mobile phase selection;

[0025] Figure 2 Chromatograms of sword bean medicinal materials at different wavelengths;

[0026] Figure 3 Column temperature investigation;

[0027] Figure 4 Flow rate investigation;

[0028] Figure 5 Injection volume inspection;

[0029] Figure 6 Delayed inspection;

[0030] Figure 7 Investigation of extraction solvents;

[0031] Figure 8 Extraction time investigation;

[0032] Figure 9 Sampling volume inspection;

[0033] Figure 10 Investigation of extraction methods;

[0034] Figure 11 Contrast with sword bean control medicinal material;

[0035] Figure 12 Surveys using different instruments;

[0036] Figure 13 Investigation of different chromatographic columns;

[0037] Figure 14 Characteristic spectrum of sword bean medicinal material (where S1-S21 are: DD01-DD21);

[0038] Figure 15 Comparative characteristic spectrum of sword bean medicinal materials;

[0039] Figure 16 Comparison of seed characteristic maps of Jack Bean and Glehnia littoralis. DETAILED DESCRIPTION

[0040] Example 1 Characteristic Spectrum Identification Method of Sword Beans and Their Counterfeits of the Present Invention

[0041] 1. Materials, reagents, and instruments

[0042] High performance liquid chromatograph: Agilent high performance liquid chromatograph, Thermo Fisher high performance liquid chromatograph, Waters high performance liquid chromatograph;

[0043] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0044] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0045] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0046] Chromatographic columns: DIKMAl C18 2.1*150mm, 1.8μm; Shimadzu C18 2.1*150mm, 1.8μm; Waters C18 2.1×150mm, 1.8μm.

[0047] 2. Reagents and test drugs

[0048] Acetonitrile (Sigma-Aldrich, chromatographic grade); phosphoric acid (chromatographic grade); and ultrapure water.

[0049] Cona bean (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTYD001301) and rutin (China Food and Drug Administration, batch number: 100080-202012, purity: 91.6%)

[0050] Sword bean medicinal material batch number: DD01~DD21.

[0051] 3. Characteristic spectrum determination method

[0052] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length, 150 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate, 0.2 ml / min; column temperature, 25°C; detection wavelength, 340 nm. The number of theoretical plates, calculated based on the rutin peak, must be no less than 5000.

[0053]

[0054] Preparation of Reference Solution: Take 1 g of the Cona bean reference medicinal material and place it in a stoppered Erlenmeyer flask. Add 50 ml of water and boil for 30 minutes. Let cool, filter, and evaporate the filtrate to dryness. Dissolve the residue in 25 ml of 80% methanol. Filter and use the filtrate as the reference medicinal material solution. Separately, accurately weigh an appropriate amount of rutin reference substance and add 80% methanol to a solution containing 20 μg per ml. This will serve as the reference substance solution.

[0055] Preparation of the test solution: Take 1.0 g of sword bean medicinal material powder (passed through No. 4 sieve), place it in a stoppered conical flask, add 25 ml of 80% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, filter, and take the filtrate to obtain the product.

[0056] Determination method: Accurately aspirate 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0057] 4. Chromatographic conditions and system suitability test

[0058] 4.1 Mobile phase selection

[0059] Based on the experimental conditions proposed above, the separation effect of acetonitrile-0.1% phosphoric acid solution mobile phase was investigated. Figure 1 .

[0060] The results showed that when the mobile phase was acetonitrile-0.1% phosphoric acid solution, the chromatographic peak baseline was relatively stable and there were more chromatographic peaks. Therefore, acetonitrile-0.1% phosphoric acid solution gradient elution was used as the mobile phase for the characteristic spectrum determination method of sword bean medicinal material.

[0061] 4.2 Wavelength Selection

[0062] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 254 nm, 280 nm, 310 nm, 340 nm, and 360 nm were extracted respectively. Figure 2 .

[0063] The results showed that the chromatographic peak information content was larger and the chromatogram baseline was more stable when the detection wavelength was 340nm-360nm.

[0064] 4.3 Column temperature investigation

[0065] Based on the experimental conditions proposed above, the column temperatures of 20℃, 25℃, 30℃ and 35℃ were investigated respectively. Figure 3 , Table 1.

[0066] Table 1 Column temperature investigation-relative retention time

[0067]

[0068] The results showed that when the column temperature was 20℃, 25℃, 30℃ and 35℃, the chromatogram peak shape was relatively symmetrical, the separation was good and the peak extraction was more complete.

[0069] 4.4 Flow rate investigation

[0070] Based on the experimental conditions proposed above, the flow rates of 0.20ml / min, 0.25ml / min and 0.30ml / min were investigated respectively. Figure 4 , Table 2.

[0071] Table 2 Flow rate investigation-relative retention time

[0072]

[0073] The results showed that the chromatographic peak shapes were good and the separation was moderate when the flow rates were 0.20ml / min, 0.25ml / min and 0.30ml / min.

[0074] 4.5 Sample injection volume investigation

[0075] Based on the experimental conditions proposed above, the injection volumes of 1μl, 2μl and 3μl were investigated respectively. Figure 5 , Table 3.

[0076] Table 3 Injection volume investigation-relative retention time

[0077]

[0078] The results showed that the chromatographic peak shape was good when the injection volume was 1-3 μl.

[0079] 4.6 Latency Investigation

[0080] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Figure 6 .

[0081] The results showed that the sample had basically no chromatographic peak after 50 minutes, so the sample detection time was set to 50 minutes.

[0082] In summary, the chromatographic conditions and system suitability testing for the characteristic spectrum of Jack Bean (canavalia) are as follows: octadecylsilane bonded silica gel as the filler (column length, 150 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; flow rate, 0.2 ml / min; column temperature, 25°C; detection wavelength, 340 nm. The number of theoretical plates, calculated based on the rutin peak, must be no less than 5000.

[0083]

[0084] 5. Preparation of test solution

[0085] 5.1 Extraction solvent investigation

[0086] Take 1.0 g of Jack Bean powder (passed through a No. 4 sieve) (batch number: DD21) and place it in a stoppered conical flask. The extraction solvents for the test sample were 25 ml each of water, methanol, 80% ethanol, 80% methanol, 50% methanol, and 30% methanol. Seal the flask tightly and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, and take the filtrate to obtain the product. Figure 7 .

[0087] The results showed that when the extraction solvent was methanol at different concentrations, the peak shapes of the characteristic peaks were good and the separation was moderate.

[0088] 5.2 Extraction time investigation

[0089] Take 1.0 g of Jack Bean powder (passed through a No. 4 sieve) (batch number: DD21), place it in a stoppered conical flask, add 25 ml of 80% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 8 .

[0090] The results showed that the peak shape and separation of the chromatograms were better when the extraction time was 20, 30 and 40 minutes.

[0091] 5.3 Sampling volume investigation

[0092] Take 0.5g, 1.0g and 1.5g of Jack Bean powder (passed through No. 4 sieve) (batch number: DD21) respectively, add 25ml of 80% methanol, place in a stoppered conical flask, seal tightly, and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes. Let cool, shake well, filter, and take the filtrate to obtain. Figure 9 .

[0093] The results showed that when the sampling amount was 0.5g, 1.0g and 1.5g, the peak shape and separation of each chromatographic peak were good.

[0094] 5.4 Investigation of extraction methods

[0095] Take 1.0g of sword bean powder (passed through No. 4 sieve) (batch number: DD21), place it in a stoppered conical flask, add 25ml of 80% methanol, seal it tightly, and ultrasonically treat it (power 600W, frequency 40kHz), reflux it for 30 minutes, let it cool, filter it, and take the filtrate to obtain the product; take another 1.0g of sword bean powder, place it in a stoppered conical flask, add 50ml of water, decoct it for 30 minutes, filter it, evaporate the filtrate to dryness, add 25ml of 80% methanol to the residue, seal it tightly, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 10 .

[0096] The results showed that there was little difference between the different extraction methods, and ultrasonic treatment was convenient and simple, so ultrasonic extraction was tentatively selected as the extraction method.

[0097] In summary, the preparation method of the test solution of the characteristic spectrum of the sword bean medicinal material is tentatively determined as follows: take 1.0 g of the sword bean medicinal material powder (passed through a No. 4 sieve), place it in a stoppered conical flask, add 25 ml of 80% methanol, seal it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it evenly, filter it, and take the filtrate to obtain it.

[0098] The preparation method of the test sample is simple and has a wide selection range. The extraction solvent of the method is methanol of different concentrations, the extraction time is 20-40 minutes, the sampling amount is 0.5g-1.5g, and the extraction method is ultrasound, reflux, decoction + ultrasound, and can achieve effective separation under the conditions.

[0099] 6. Methodological Investigation

[0100] 6.1 Chromatographic Peak Identification

[0101] Preparation of the test solution: Take 1.0 g of sword bean powder (passed through a No. 4 sieve), place it in a stoppered conical flask, add 25 ml of 80% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Cool it, filter it, and take the filtrate to obtain the product.

[0102] Preparation of control medicinal material solution: Take 1 g of sword bean control medicinal material, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 80% methanol to dissolve the residue, filter, and take the filtrate as the control medicinal material reference solution.

[0103] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution lacking the sword bean medicinal material.

[0104] Compare the peaks of the characteristic spectrum of the reference medicinal material, sword bean. Figure 11 .

[0105] The results showed that peak 4 was rutin. In the following methodological investigation, eight characteristic peaks in the sample were investigated.

[0106] 6.2 Precision test

[0107] Take the test solution of Jack Bean (Batch No. DD21) and inject 2 μl of the solution six times according to the proposed experimental method. Calculate the relative retention time and peak area of ​​each characteristic peak. See Table 4.

[0108] Table 4 Precision investigation-retention time

[0109]

[0110] The results showed that the RSD of the relative retention time of each characteristic peak of the sample was 0.05% to 0.37%, and the instrument had good precision.

[0111] 6.3 Repeatability Study

[0112] Six samples of Cona bean (Batch No. DD21) were prepared and assayed according to the proposed experimental method. See Table 5.

[0113] Table 5 Repeatability study - relative retention time

[0114]

[0115] The results showed that the RSD of the relative retention time of the six samples was 0.05% to 0.29%, indicating that the method had good repeatability.

[0116] 6.4 Intermediate precision study

[0117] 6.4.1 Investigation of different instruments

[0118] Based on the experimental conditions proposed above, two portions of Jack Bean (Batch No.: DD21) were weighed to prepare test solutions, which were then measured on Waters, Thermo Fisher, and Agilent HPLC instruments, respectively. See Table 6. Figure 12 .

[0119] Table 6 Instrument durability investigation - relative retention time

[0120]

[0121] The results showed that when the three instruments were used to detect the test samples, the RSDs of the relative retention times of the characteristic peaks were 0.53% to 2.30%, indicating that the instruments had good durability.

[0122] 6.4.2 Inspection by different personnel and time

[0123] Based on the experimental conditions proposed above, two samples of Jack Bean (Batch No.: DD21) were weighed by different individuals (A and B) at different times (T1 and T2) to prepare test samples for determination (see Table 7).

[0124] Table 7 Personnel and time inspection-relative retention time

[0125]

[0126] The results showed that the RSDs of the relative retention times of the characteristic peaks were 0.06% to 0.38%, indicating that the method was stable.

[0127] 6.5 Durability inspection

[0128] 6.5.1 Column Durability Assessment

[0129] Based on the experimental conditions proposed above, the chromatographic columns DIKMAC18 2.1*150mm, 1.8μm; Shimadzu C18 2.1*150mm, 1.8μm; and Waters C18 2.1×150mm, 1.8μm were analyzed and investigated. The results are shown in Table 8. Figure 13 .

[0130] Table 8 Column durability investigation - relative retention time

[0131]

[0132] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 1.83% and 4.25% when the three chromatographic columns were used to detect the samples, indicating that the chromatographic columns had good durability.

[0133] 6.5.2 Stability investigation

[0134] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h. See Table 9.

[0135] Table 9 Stability Study-Retention Time

[0136]

[0137] The results showed that the RSD of the characteristic peak retention time was between 0.03% and 0.35%, and the sample solution was stable within 24 hours.

[0138] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above eight characteristic peaks were included in the subsequent investigations.

[0139] 7. Verification of the characteristic spectrum of sword bean medicinal materials

[0140] The sword bean medicinal material was tested and the relative retention time and relative peak area were calculated. The results are shown in Figure 14 , Table 10.

[0141] Table 10 Relative retention time of characteristic spectra of sword bean medicinal materials

[0142]

[0143]

[0144] Based on the principle that the relative retention time is stable and all batches of samples can be detected with relatively high peaks, a total of 8 peaks with good reproducibility were selected as characteristic peaks.

[0145] Final regulations: The test sample chromatogram should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the rutin reference peak is the S peak, and the relative retention times of the remaining peaks to the S peak are calculated; their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.47 (peak 1), 0.88 (peak 2), 0.96 (peak 3), 1.52 (peak 5), 1.56 (peak 6), 1.58 (peak 7), and 1.61 (peak 8).

[0146] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) was used to synthesize 21 batches of Jack Bean medicinal materials, and a reference characteristic spectrum of Jack Bean medicinal materials was established. Figure 15 .

[0147] 8. Comparison of characteristic patterns of sword bean medicinal materials and Hedera oleifera seeds

[0148] This method was used to test the medicinal materials of sword bean and seeds of Hedera helichrysum. The results are shown in Figure 16 .

[0149] 8.1 Materials, reagents, and instruments

[0150] High performance liquid chromatograph: Agilent high performance liquid chromatograph;

[0151] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0152] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0153] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0154] Chromatographic column: DIKMAl C18 2.1*150mm, 1.8μm;

[0155] 8.2 Reagents and test drugs

[0156] Acetonitrile (Sigma-Aldrich, chromatographic grade); phosphoric acid (chromatographic grade); and ultrapure water.

[0157] Jack bean medicinal material batch number: DD21;

[0158] Evergreen jasmine seed batch number: CCYMT01.

[0159] 8.3 Characteristic spectrum determination method

[0160] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length, 150 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate, 0.2 ml / min; column temperature, 25°C; detection wavelength, 340 nm. The number of theoretical plates, calculated based on the rutin peak, must be no less than 5000.

[0161]

[0162] Preparation of test solution: Take 1.0 g of the powder of sword bean and the powder of Hedera chinensis seeds (passed through No. 4 sieve), place them in a stoppered conical flask, add 25 ml of 80% methanol respectively, stopper it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Cool it, filter it, and take the filtrate to obtain the product.

[0163] Determination method: Accurately aspirate 2μl of the test solution respectively, inject it into the liquid chromatograph, and determine it.

[0164] The results showed that there were eight characteristic peaks in the jack bean. The retention times of Peaks 1-8 were 11.846 min, 21.962 min, 24.020 min, 25.023 min, 38.115 min, 38.991 min, 39.534 min, and 40.320 min, respectively. Peak 4 was designated as the S peak, and the relative retention times of the remaining peaks relative to the S peak were 0.47 (Peak 1), 0.88 (Peak 2), 0.96 (Peak 3), 1.52 (Peak 5), 1.56 (Peak 6), 1.58 (Peak 7), and 1.61 (Peak 8), respectively. All relative retention times were within ±10% of the specified values. However, no corresponding chromatographic peaks were detected in Hedera oleifera seeds at the retention times of Peaks 1-8. Therefore, Peaks 1-8 can be used as distinguishing points between Jack bean and its counterfeit Hedera oleifera seeds.

Claims

1. A method for identifying sword beans and their counterfeits using characteristic patterns, characterized in that: It includes the following steps: a. Pre-treatment of samples to be tested; b. The liquid to be tested is measured by high performance liquid chromatography to obtain HPLC characteristic spectrum of sword bean and its preparation; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an acetonitrile solution, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the gradient elution is performed; The gradient elution is specifically as follows: 0-12 min, phase A: 10→14%, phase B: 90→86%; 12-22 min, phase A: 14% → 16%, phase B: 86% → 84%; 22-25 min, phase A: 16% → 18%, phase B: 84% → 82%; 25-45 min, phase A: 18% → 30%, phase B: 82% → 70%; 45-50 min, phase A: 30%, phase B: 70%; c. Compare the characteristic peaks. The relative retention time of the characteristic peaks is: Peak 1: 0.47, peak 2: 0.88, peak 3: 0.96, peak 5: 1.52, peak 6: 1.56, peak 7: 1.58, peak 8: 1.61, the relative retention times are within ±10% of the specified values; If the relative retention time of the sample to be tested corresponds to the above-mentioned characteristic peak, it is a genuine sword bean; if the relative retention time does not correspond to the above-mentioned characteristic peak, it is a counterfeit sword bean.

2. The method for identifying sword beans and counterfeits thereof using characteristic patterns according to claim 1, wherein: The pretreatment method of the sample to be tested in step a is: Take the sample to be tested, add methanol, ultrasonically treat, cool, filter, and take the filtrate.

3. The method for identifying sword beans and counterfeits thereof using characteristic patterns according to claim 2, wherein: The methanol concentration is 80%, and the ultrasonic treatment conditions are: power 600W, frequency 40kHz; and treatment time 30 minutes.

4. The method for identifying sword beans and counterfeits thereof using characteristic patterns according to claim 1, wherein: The chromatographic column is C18 150×2.1mm 1.8μm; the column temperature is 20-35°C; the theoretical plate number calculated based on rutin should be no less than 5000; the mobile phase flow rate is 0.2-0.3mL / min; the injection volume is 1-3μL; and the detection wavelength is 340-360nm.

5. The method for identifying sword beans and counterfeits thereof using characteristic patterns according to claim 4, wherein: The column temperature is 25° C.; the mobile phase flow rate is 0.2 mL / min; the injection volume is 2 μL; and the detection wavelength is 340 nm.

6. The method for identifying sword beans and counterfeits thereof according to any one of claims 1 to 5, wherein: The fake sword bean is: Mucuna sempervirens Hemsl. seeds.

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