Characteristic map construction and detection of Chinese yam and identification method of its processed or counterfeit products

Abstract

The present invention relates to a method for constructing and detecting a characteristic spectrum of Chinese yam, as well as identifying processed or counterfeit products thereof. The method for constructing a characteristic spectrum of Chinese yam comprises the following steps: taking a Chinese yam sample to be tested and preparing a test solution; performing ultra-high performance liquid chromatography analysis on the test solution to construct a characteristic spectrum of the Chinese yam; the chromatographic conditions include: methanol as mobile phase A, ammonium acetate aqueous solution as mobile phase B, and gradient elution, wherein the volume percentage of mobile phase A in the elution sequence is: 0% from 0 to 10 minutes; 0% to 3% from 10 to 22 minutes; 3% to 12% from 22 to 30 minutes; 12% to 30% from 30 to 40 minutes; and 30% to 65% from 40 to 45 minutes. The method has the advantages of being time-efficient, simple to operate, rich in chromatographic information, highly characteristic, and reproducible. It can distinguish Chinese yam from stir-fried Chinese yam with braised bran based on the difference in the number of characteristic peaks, and can distinguish Chinese yam from counterfeit products based on the relative peak areas of the characteristic peaks.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine identification, and in particular to a method for constructing and detecting a characteristic spectrum of yam and identifying its processed products and counterfeit products. Background Art

[0002] Chinese yam, listed in Part I of the 2020 edition of the Chinese Pharmacopoeia, is derived from the dried rhizome of Dioscorea opposita Thunb. (Dioscorea opposita Thunb.), a plant of the Dioscoreaceae family. Chinese yam is sweet and neutral in nature. It enters the spleen, lung, and kidney meridians. It nourishes the spleen and stomach, promotes fluid production and benefits the lungs, and tonifies the kidneys and astringes semen. It is used for spleen deficiency, poor appetite, chronic diarrhea, wheezing and coughing due to lung deficiency, spermatorrhea due to kidney deficiency, leucorrhea, frequent urination, and thirst due to deficiency heat. Stir-fried Chinese yam with bran nourishes the spleen and stomach. It is used for spleen deficiency, poor appetite, diarrhea, loose stools, and excessive leucorrhea.

[0003] The current pharmacopoeia specifies that yam is derived from the dried rhizome of Dioscorea opposita Thunb., a member of the Dioscoreaceae family. However, many species of the same genus can resemble each other after slicing, making identification of their origin difficult. Common counterfeit yam products include the dried rhizome of Dioscarea persimilis Prain et Burkill, also of the Dioscoreaceae family, known as "Guangshanyao." The tuberous root of Manihot esculenta Crantz, a member of the Euphorbiaceae family, is often sliced ​​horizontally and obliquely, resembling yam slices. The tuber of Dioscorea alata L., also of the Dioscoreaceae family, is used in some regions as "Huaishanyao." While there are reports on methods for distinguishing yam from its counterfeit counterparts, most rely on appearance, microscopic features, physicochemical parameters, DNA barcoding, and near-infrared technology, there are no reports specifically addressing the composition of the yam.

[0004] According to modern medical research, yam is rich in saponins, mucilage, glycoprotein, mannan, phytic acid, allantoin, yamin, choline, dopamine, crude fiber, pectin, amylase and various trace elements. Yam has the functions of regulating or enhancing immune function, anti-fatigue, anti-liver damage, anti-tumor, lowering blood sugar, lowering blood lipids, anti-oxidation, anti-aging, anti-inflammation, antibacterial, strengthening the spleen and stomach, and promoting digestion.

[0005] Currently, there have been studies on the quality standards of Chinese yam or braised Chinese yam. For example, Li Lailing conducted a fingerprint study on Chinese yam slices using HPLC, using an Ultimate XB-C18 column with a linear gradient elution (0-50 min, 99%-80% water, 1%-20% methanol), a flow rate of 1.0 ml / min, a column temperature of 30°C, and a detection wavelength of 258 nm. The generated fingerprints showed that the overall appearance of the main chromatographic peaks in the fingerprints of 10 batches of Chinese yam slices was essentially consistent, with all having the same chromatographic characteristic peaks. Wang Hui et al. established a method for characterizing the characteristic spectra of Chinese yam and braised Chinese yam slices. The results showed that the characteristic spectra of both Chinese yam and braised Chinese yam slices showed 10 characteristic peaks, and there were differences between the yam before and after frying. However, only the allantoin peak in the characteristic spectra was attributed, and this component elutes early, almost simultaneously with the solvent peak. Wang Bijun et al. established characteristic spectra for Chinese yam and braised Chinese yam with bran. They identified five common peaks across 10 batches of raw Chinese yam and eight common peaks across processed Chinese yam. They identified only adenosine, but did not investigate common Chinese yam counterfeits. Peng Yan et al. demonstrated significant differences in the Raman fingerprints of Chinese yam before and after braised Chinese yam, indicating changes in the main component (starch) of Chinese yam after braised Chinese yam. Du Xingang et al. developed a thin-layer chromatography method for distinguishing Chinese yam formula granules from braised Chinese yam formula granules with bran. However, the TLC analysis revealed two distinct spots, with unclear distinguishing spots and their components not clearly defined.

[0006] Therefore, it is urgent to provide a method that is fast and effective and can distinguish Chinese yam and its processed products and counterfeit products. Summary of the Invention

[0007] Based on this, the present invention provides a method for constructing a characteristic spectrum of yam, which attributes 6 components: cytidine, uridine, adenine, guanosine, tryptophan, and adenosine. It has the advantages of short time consumption, simple operation, rich spectrum information, strong characteristic and good reproducibility. It can identify yam and fried yam with bran according to the difference in the number of characteristic peaks, and identify yam and its counterfeit according to the relative peak area of ​​the characteristic peaks.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for constructing a characteristic spectrum of Chinese yam comprises the following steps:

[0010] Take the yam sample to be tested and prepare the test solution;

[0011] The test solution is subjected to ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of yam;

[0012] Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include:

[0013] Gradient elution with a mobile phase was used, wherein the mobile phase included mobile phase A and mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution;

[0014] The procedure of the gradient elution is:

[0015] From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0016] From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%;

[0017] From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%;

[0018] From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%;

[0019] From 40 min to 45 min, the volume percentage of mobile phase A changed from 30% to 65%, and the volume percentage of mobile phase B changed from 70% to 35%.

[0020] In one embodiment, the concentration of ammonium acetate in the aqueous ammonium acetate solution is 0.02 mol / L to 0.03 mol / L.

[0021] In one embodiment, the chromatographic conditions of ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 250nm to 270nm.

[0022] In one embodiment, the solvent in the test solution is selected from an alcohol-containing aqueous solution and a mixture of one or two of water. Further, the solvent in the test solution is selected from a methanol-containing aqueous solution and a mixture of one or two of water.

[0023] In one embodiment, preparing the test solution comprises the following steps:

[0024] The yam sample to be tested is mixed with an alcohol-containing aqueous solution, subjected to ultrasonic extraction, filtered, and the filtrate is collected.

[0025] In one embodiment, the method for constructing a characteristic spectrum of yam further includes the steps of preparing a control medicinal material reference solution and / or a mixed control material reference solution, and performing ultra-performance liquid chromatography analysis on the control medicinal material reference solution and / or the mixed control material reference solution.

[0026] In one embodiment, the solvents in the control medicinal material reference solution and the mixed reference substance solution are independently selected from one or a mixture of an alcohol-containing aqueous solution and water. Furthermore, the solvents in the control medicinal material reference solution and the mixed reference substance solution are independently selected from one or a mixture of an aqueous methanol solution and water.

[0027] In one embodiment, preparing a control medicinal material reference solution comprises the following steps:

[0028] The yam sample is mixed with an alcohol-containing aqueous solution, heated to reflux, filtered, and the filtrate is collected.

[0029] In one embodiment, preparing a mixed reference substance solution comprises the following steps:

[0030] Cytidine, uridine, adenine, guanosine, tryptophan, adenosine and a methanol aqueous solution with a methanol volume percentage of 5% to 20% are mixed.

[0031] In one embodiment, the yam sample to be tested is selected from yam medicinal materials or yam slices fried with bran.

[0032] The present invention also provides a method for detecting yam, comprising the following steps:

[0033] Taking a yam sample to be tested and preparing a yam sample solution to be tested;

[0034] The yam sample solution to be tested is subjected to ultra-high performance liquid chromatography analysis, and the obtained chromatogram is compared with the characteristic spectrum of yam constructed by the method for constructing the characteristic spectrum of yam as described above;

[0035] Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include:

[0036] Gradient elution with a mobile phase was used, wherein the mobile phase included mobile phase A and mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution;

[0037] The procedure of the gradient elution is:

[0038] From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0039] From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%;

[0040] From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%;

[0041] From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%;

[0042] From 40 min to 45 min, the volume percentage of mobile phase A changed from 30% to 65%, and the volume percentage of mobile phase B changed from 70% to 35%.

[0043] In one embodiment, in the ammonium acetate aqueous solution used in the yam detection method, the concentration of ammonium acetate is 0.02 mol / L to 0.03 mol / L.

[0044] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography analysis used in the yam detection method also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 250nm to 270nm.

[0045] In one embodiment, the solvent in the yam sample solution to be tested is selected from one or a mixture of an alcohol-containing aqueous solution and water. Further, the solvent in the yam sample solution to be tested is selected from one or a mixture of a methanol-containing aqueous solution and water.

[0046] In one embodiment, preparing the yam sample solution to be tested comprises the following steps:

[0047] The yam sample to be tested is mixed with an alcohol-containing aqueous solution, subjected to ultrasonic extraction, filtered, and the filtrate is collected.

[0048] In one embodiment, the method for detecting yam further includes the steps of preparing a control medicinal material reference solution and / or a mixed control reference solution, and performing ultra-performance liquid chromatography analysis on the control medicinal material reference solution and / or the mixed control reference solution.

[0049] In one embodiment, in the method for detecting Chinese yam, the solvents in the control medicinal material reference solution and the mixed reference substance solution are independently selected from one or a mixture of an alcohol-containing aqueous solution and water. Furthermore, in the method for detecting Chinese yam, the solvents in the control medicinal material reference solution and the mixed reference substance solution are independently selected from one or a mixture of an aqueous methanol solution and water.

[0050] In one embodiment, in the method for detecting yam, preparing a control medicinal material reference solution comprises the following steps:

[0051] The yam sample is mixed with an alcohol-containing aqueous solution, heated to reflux, filtered, and the filtrate is collected.

[0052] In one embodiment, in the method for detecting yam, preparing a mixed reference substance solution comprises the following steps:

[0053] Cytidine, uridine, adenine, guanosine, tryptophan, adenosine and a methanol aqueous solution with a methanol volume percentage of 5% to 20% are mixed.

[0054] The present invention also provides a method for identifying processed or counterfeit yam products, comprising the following steps:

[0055] Taking a sample to be tested and preparing a sample solution to be tested;

[0056] The sample solution is subjected to ultra-high performance liquid chromatography analysis, and the obtained chromatogram is compared with the characteristic spectrum of yam constructed by the characteristic spectrum construction method of yam as described above;

[0057] Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include:

[0058] Gradient elution with a mobile phase was used, wherein the mobile phase included mobile phase A and mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution;

[0059] The procedure of the gradient elution is:

[0060] From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0061] From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%;

[0062] From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%;

[0063] From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%;

[0064] From 40 min to 45 min, the volume percentage of mobile phase A changed from 30% to 65%, and the volume percentage of mobile phase B changed from 70% to 35%.

[0065] In one embodiment, the concentration of ammonium acetate in the aqueous solution of ammonium acetate used in the method for identifying processed yam or counterfeit yam is 0.02 mol / L to 0.03 mol / L.

[0066] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography analysis used in the method for identifying processed or counterfeit yam also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 250nm to 270nm.

[0067] Compared with the prior art, the method for constructing the characteristic spectrum of yam of the present invention has the following beneficial effects:

[0068] The present invention uses ultra-high performance liquid chromatography to establish a characteristic spectrum for Chinese yam. By selecting methanol and ammonium acetate aqueous solution as the mobile phases and limiting the elution method to gradient elution, the resulting characteristic spectrum for Chinese yam is applicable to both Chinese yam and Chinese yam slices. It can distinguish Chinese yam from stir-fried Chinese yam with braised bran based on the difference in the number of characteristic peaks, and distinguish Chinese yam from counterfeit products based on the relative peak areas of the characteristic peaks. Furthermore, the characteristic spectrum for Chinese yam constructed in the present invention has six common peaks, is rich in information, highly characteristic, and has good reproducibility. It also offers the advantages of simple operation and reduced processing time.

[0069] Furthermore, the characteristic spectrum construction method of yam of the present invention can distinguish and identify yam from its processed products (such as fried yam with bran) or counterfeit products (such as ginseng yam, Guang yam or cassava). BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Overlay diagram of characteristic spectra of 19 batches of yam medicinal materials provided by the present invention;

[0071] Figure 2 The comparative characteristic spectrum of the Chinese yam medicinal material provided by the present invention;

[0072] Figure 3 Characteristic spectrum of Chinese yam reference medicinal material provided by the present invention;

[0073] Figure 4 The characteristic spectrum overlay of 19 batches of fried yam slices provided by the present invention;

[0074] Figure 5 The comparative characteristic spectrum of the fried yam slices with bran provided by the present invention;

[0075] Figure 6 Comparison of characteristic spectra of Chinese yam medicinal material and bran-fried Chinese yam slices provided by the present invention;

[0076] Figure 7 This is an overlay of characteristic spectra of three counterfeit yam products provided by the present invention;

[0077] Figure 8 The chromatogram of the yam medicinal material obtained by isocratic elution with methanol and water provided by the present invention. DETAILED DESCRIPTION

[0078] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0080] In the present invention, m% methanol aqueous solution refers to a methanol aqueous solution with a methanol volume percentage of m%, such as a 10% methanol aqueous solution refers to a methanol aqueous solution with a methanol volume percentage of 10%.

[0081] Currently, the 2020 edition of the Chinese Pharmacopoeia only lists properties, identification, inspection, and extracts for Chinese yam, without specifying the content of its indicative components or its characteristic profile. The properties section is of limited reference value for distinguishing decoction pieces that have changed their morphology. The identification methods reported in the literature for Chinese yam and its counterfeits have certain limitations.

[0082] Compared with previous research reports, the present invention uses the UPLC method to establish characteristic spectrum detection methods for yam medicinal materials and fried yam slices with bran, and attributes six components: cytidine, uridine, adenine, guanosine, tryptophan, and adenosine. Yam and fried yam with bran are distinguished based on the difference in the number of characteristic peaks, and yam and its counterfeit are distinguished based on the relative peak areas of the characteristic peaks.

[0083] A method for constructing a characteristic spectrum of Chinese yam comprises the following steps:

[0084] Take the yam sample to be tested and prepare the test solution;

[0085] The test solution is subjected to ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of yam;

[0086] Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include:

[0087] Gradient elution with a mobile phase was used, wherein the mobile phase included mobile phase A and mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution;

[0088] The procedure of the gradient elution is:

[0089] From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0090] From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%;

[0091] From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%;

[0092] From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%;

[0093] From 40 min to 45 min, the volume percentage of mobile phase A changed from 30% to 65%, and the volume percentage of mobile phase B changed from 70% to 35%.

[0094] In one embodiment, the concentration of ammonium acetate in the aqueous solution of ammonium acetate is 0.02 mol / L to 0.03 mol / L, including but not limited to 0.02 mol / L, 0.021 mol / L, 0.022 mol / L, 0.023 mol / L, 0.024 mol / L, 0.025 mol / L, 0.026 mol / L, 0.027 mol / L, 0.028 mol / L, 0.029 mol / L and 0.03 mol / L.

[0095] In one embodiment, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 250nm to 270nm.

[0096] Preferably, the chromatographic column is Waters HSS T3 (2.1 mm×150 mm, 1.8 μm).

[0097] Preferably, the column temperature is 30° C.; the flow rate is 0.3 mL / min; and the wavelength is 258 nm.

[0098] In one embodiment, the chromatographic conditions of ultra-high performance liquid chromatography analysis further include: an injection volume of 2 μL.

[0099] In one embodiment, the solvent in the test solution is selected from an alcohol-containing aqueous solution and a mixture of one or both of water. Further, the solvent in the test solution is selected from a methanol-water solution and a mixture of one or both of water. Furthermore, the solvent in the test solution is selected from a methanol-water solution.

[0100] In one embodiment, preparing the test solution comprises the following steps:

[0101] The yam sample to be tested is mixed with an alcohol-containing aqueous solution, subjected to ultrasonic extraction, filtered, and the filtrate is collected.

[0102] In one embodiment, the conditions for ultrasonic extraction include: power of 250W to 350W, frequency of 30kHz to 50kHz, time of 30min to 60min, and temperature of 25°C to 35°C.

[0103] Furthermore, preparing the test solution comprises the following steps:

[0104] Take the test sample powder (pass through No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 10% methanol aqueous solution, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, take it out, cool, weigh it again, make up the lost weight with 10% methanol, shake well, filter, and take the filtrate to obtain.

[0105] In one embodiment, the method for constructing a characteristic spectrum of yam further includes the steps of preparing a control medicinal material reference solution and / or a mixed control material reference solution, and performing ultra-performance liquid chromatography analysis on the control medicinal material reference solution and / or the mixed control material reference solution.

[0106] In one embodiment, the solvent in the control medicinal material reference solution is selected from one or a mixture of an alcohol-containing aqueous solution and water. Furthermore, the solvent in the control medicinal material reference solution is selected from one or a mixture of a methanol-containing aqueous solution and water.

[0107] In one embodiment, preparing a control medicinal material reference solution comprises the following steps:

[0108] The yam sample is mixed with an alcohol-containing aqueous solution, heated to reflux, filtered, and the filtrate is collected.

[0109] Furthermore, preparing the control medicinal material reference solution includes the following steps:

[0110] Take the Chinese yam control medicinal material, place it in a stoppered conical flask, add 10% methanol aqueous solution, heat and reflux for 30 minutes, take it out, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0111] In one embodiment, the solvent in the mixed reference substance solution is selected from a mixture of one or both of an alcoholic aqueous solution and water. Further, the solvent in the mixed reference substance solution is selected from a mixture of one or both of a methanol aqueous solution and water.

[0112] In one embodiment, preparing a mixed reference substance solution comprises the following steps:

[0113] Cytidine, uridine, adenine, guanosine, tryptophan, adenosine and a methanol aqueous solution with a methanol volume percentage of 5% to 20% are mixed.

[0114] Furthermore, preparing the mixed reference substance solution comprises the following steps:

[0115] Take appropriate amounts of cytidine, uridine, adenine, guanosine, tryptophan and adenosine, accurately weigh them, and add 10% methanol aqueous solution to prepare a mixed reference substance solution.

[0116] In one embodiment, the yam sample to be tested is selected from yam medicinal materials or yam slices fried with bran.

[0117] In one of the more preferred embodiments, the method for constructing the characteristic spectrum of Chinese yam of the present invention comprises the following steps:

[0118] Take Chinese yam control medicinal material and prepare control medicinal material reference solution;

[0119] Prepare a mixed reference solution by taking cytidine, uridine, adenine, guanosine, tryptophan, and adenosine;

[0120] Take the yam sample to be tested and prepare the test solution;

[0121] The control medicinal material reference solution, the mixed reference substance solution and the test sample solution are subjected to ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of yam;

[0122] Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include:

[0123] Gradient elution with a mobile phase was used, wherein the mobile phase included mobile phase A and mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution;

[0124] The procedure of the gradient elution is:

[0125] From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0126] From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%;

[0127] From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%;

[0128] From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%;

[0129] From 40 min to 45 min, the volume percentage of mobile phase A changed from 30% to 65%, and the volume percentage of mobile phase B changed from 70% to 35%.

[0130] In one embodiment, the method for constructing the characteristic spectrum of Chinese yam comprises the following steps:

[0131] (1) Preparation of reference solution:

[0132] Take about 1g of Chinese yam control medicinal material, place it in a stoppered conical flask, add 25ml of 10% methanol aqueous solution, heat and reflux for 30 minutes, take it out, let it cool, shake it well, filter it, and take the filtrate as the control medicinal material reference solution;

[0133] Separately, take appropriate amounts of cytidine, uridine, adenine, guanosine, tryptophan and adenosine, accurately weigh them, and add 10% methanol aqueous solution to prepare a mixed solution containing 8 μg-15 μg of each reference substance per 1 ml, which serves as the reference substance solution.

[0134] (2) Preparation of test solution:

[0135] Take about 0.5 g of the sample powder to be tested (passed through a No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 10% methanol, weigh it, and ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 10% methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain it.

[0136] (3) Chromatographic conditions and system suitability test:

[0137] Octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); methanol was used as mobile phase A, and 0.03 mol / L ammonium acetate solution was used as mobile phase B, and gradient elution was performed as specified in Table 1 below; the flow rate was 0.3 ml per minute; the column temperature was 30°C; the detection wavelength was 258 nm, and the number of theoretical plates calculated based on the guanosine peak should be no less than 5000.

[0138] Table 1 Gradient elution table

[0139]

[0140] (4) Determination method: Accurately aspirate 2 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0141] (5) Results:

[0142] 1) The chromatographic peaks of the characteristic spectrum of yam medicinal material were assigned as follows: peak 1 is cytidine; peak 2 is uridine; peak 3 is adenine; peak 4 is guanosine; peak 5 is tryptophan; and peak 6 is adenosine.

[0143] 2) The chromatographic peaks of the characteristic spectrum of the fried yam slices with bran were attributed, which were as follows: peak 2 was cytidine; peak 3 was uridine; peak 5 was adenine; peak 7 was guanosine; peak 9 was tryptophan; and peak 11 was adenosine.

[0144] The present invention also provides a method for detecting yam, comprising the following steps:

[0145] Taking a yam sample to be tested and preparing a yam sample solution to be tested;

[0146] The yam sample solution to be tested is subjected to ultra-high performance liquid chromatography analysis, and the obtained chromatogram is compared with the characteristic spectrum of yam constructed by the method for constructing the characteristic spectrum of yam as described above;

[0147] Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include:

[0148] Gradient elution with a mobile phase was used, wherein the mobile phase included mobile phase A and mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution;

[0149] The procedure of the gradient elution is:

[0150] From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0151] From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%;

[0152] From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%;

[0153] From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%;

[0154] From 40 min to 45 min, the volume percentage of mobile phase A changed from 30% to 65%, and the volume percentage of mobile phase B changed from 70% to 35%.

[0155] In one embodiment, the ammonium acetate aqueous solution used in the yam detection method has an ammonium acetate concentration of 0.02 mol / L to 0.03 mol / L.

[0156] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography analysis used in the yam detection method also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 250nm to 270nm.

[0157] In one embodiment, the solvent in the yam sample solution to be tested is selected from one or a mixture of an alcohol-containing aqueous solution and water. Further, the solvent in the yam sample solution to be tested is selected from one or a mixture of a methanol-containing aqueous solution and water.

[0158] In one embodiment, preparing the yam sample solution to be tested comprises the following steps:

[0159] The yam sample to be tested is mixed with an alcohol-containing aqueous solution, subjected to ultrasonic extraction, filtered, and the filtrate is collected.

[0160] In one embodiment, the conditions for ultrasonic extraction include: power of 250W to 350W, frequency of 30kHz to 50kHz, time of 30min to 60min, and temperature of 25°C to 35°C.

[0161] Furthermore, preparing the yam sample solution to be tested includes the following steps:

[0162] Take the yam sample powder to be tested (passed through No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 10% methanol aqueous solution, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 10% methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain it.

[0163] In one embodiment, the method for detecting yam further includes the steps of preparing a control medicinal material reference solution and / or a mixed control reference solution, and performing ultra-performance liquid chromatography analysis on the control medicinal material reference solution and / or the mixed control reference solution.

[0164] In one embodiment, in the method for detecting yam, the solvent in the control medicinal material reference solution is selected from one or a mixture of an alcohol-containing aqueous solution and water. Furthermore, the solvent in the control medicinal material reference solution is selected from one or a mixture of a methanol-containing aqueous solution and water.

[0165] In one embodiment, in the method for detecting yam, preparing a control medicinal material reference solution comprises the following steps:

[0166] The yam sample is mixed with an alcohol-containing aqueous solution, heated to reflux, filtered, and the filtrate is collected.

[0167] Furthermore, preparing the control medicinal material reference solution includes the following steps:

[0168] Take the Chinese yam control medicinal material, place it in a stoppered conical flask, add 10% methanol aqueous solution, heat and reflux for 30 minutes, take it out, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0169] In one embodiment, in the method for detecting yam, the solvent in the mixed reference substance solution is selected from a mixture of one or both of an alcohol-containing aqueous solution and water. Furthermore, the solvent in the mixed reference substance solution is selected from a mixture of one or both of an aqueous methanol solution and water.

[0170] In one embodiment, in the method for detecting yam, preparing a mixed reference substance solution comprises the following steps:

[0171] Cytidine, uridine, adenine, guanosine, tryptophan, adenosine and a methanol aqueous solution with a methanol volume percentage of 5% to 20% are mixed.

[0172] Furthermore, preparing the mixed reference substance solution comprises the following steps:

[0173] Take appropriate amounts of cytidine, uridine, adenine, guanosine, tryptophan and adenosine, accurately weigh them, and add 10% methanol aqueous solution to prepare a mixed reference substance solution.

[0174] In one of the more preferred embodiments, the method for detecting Chinese yam of the present invention comprises the following steps:

[0175] Take Chinese yam control medicinal material and prepare control medicinal material reference solution;

[0176] Prepare a mixed reference solution by taking cytidine, uridine, adenine, guanosine, tryptophan, and adenosine;

[0177] Taking a yam sample to be tested and preparing a yam sample solution to be tested;

[0178] The yam sample solution to be tested is subjected to ultra-high performance liquid chromatography analysis, and the obtained chromatogram is compared with the characteristic spectrum of yam constructed by the method for constructing the characteristic spectrum of yam as described above;

[0179] Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include:

[0180] Gradient elution with a mobile phase was used, wherein the mobile phase included mobile phase A and mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution;

[0181] The procedure of the gradient elution is:

[0182] From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0183] From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%;

[0184] From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%;

[0185] From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%;

[0186] From 40 min to 45 min, the volume percentage of mobile phase A changed from 30% to 65%, and the volume percentage of mobile phase B changed from 70% to 35%.

[0187] The present invention also provides a method for identifying processed or counterfeit yam products, comprising the following steps:

[0188] Taking a sample to be tested and preparing a sample solution to be tested;

[0189] The sample solution is subjected to ultra-high performance liquid chromatography analysis, and the obtained chromatogram is compared with the characteristic spectrum of yam constructed by the characteristic spectrum construction method of yam as described above;

[0190] Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include:

[0191] Gradient elution with a mobile phase was used, wherein the mobile phase included mobile phase A and mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution;

[0192] The procedure of the gradient elution is:

[0193] From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0194] From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%;

[0195] From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%;

[0196] From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%;

[0197] From 40 min to 45 min, the volume percentage of mobile phase A changed from 30% to 65%, and the volume percentage of mobile phase B changed from 70% to 35%.

[0198] In one embodiment, the concentration of ammonium acetate in the aqueous solution of ammonium acetate used in the method for identifying processed yam or counterfeit yam is 0.02 mol / L to 0.03 mol / L.

[0199] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography analysis used in the method for identifying processed or counterfeit yam also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 250nm to 270nm.

[0200] The following is a further detailed description of the method for constructing the characteristic spectrum of the Fructus Nelumboe with reference to specific examples. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

[0201] Example 1

[0202] This embodiment provides a method for constructing a characteristic spectrum of yam, a detection method, and a method for identifying processed or counterfeit products, as follows:

[0203] 1.1 Instruments and reagents

[0204] Instruments: Waters ultra-high performance liquid chromatograph (Waters H-Class, Waters Corporation), Agilent ultra-high performance liquid chromatograph (Agilent 1290, Agilent); Waters HSS T3 chromatographic column (150 mm × 2.1 mm, 1.8 μm); 1 / 10,000 balance (ME204E, Mettler-Toledo); 1 / 10,000 balance (XP26, Mettler-Toledo); CNC ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instrument Co., Ltd.), constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck & Co., Ltd.).

[0205] Reagents: ethanol (Xilong Scientific Co., Ltd.) and methanol (Xilong Scientific Co., Ltd.) were of analytical grade; methanol (Merck Co., Ltd.), acetonitrile (Merck Co., Ltd.), and phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd.) were of chromatographic grade; and water was ultrapure water (prepared in the laboratory).

[0206] Test drugs: adenosine (batch number: 110879-201703, content: 99.7%, China Food and Drug Inspection Institute); uridine (batch number: 110887-201803, content: 99.50%, China Food and Drug Inspection Institute); guanosine (batch number: 111977-201501, content: 93.60%, China Food and Drug Inspection Institute); tryptophan (batch number: 140686-201904, content: 99.90%, China Food and Drug Inspection Institute); adenine (batch number: 110886-201102, content: 99.40%, China Food and Drug Inspection Institute); cytidine (batch number: wkq19011101, content: 98.72%, Sichuan Weikeqi Biotechnology Co., Ltd.).

[0207] The Chinese Pharmacopoeia (2020 edition, Part I) defines yam as the dried rhizome of Dioscorea opposita Thunb., a plant of the Dioscoreaceae family. The medicinal material used in this study was identified as the dried rhizome of Dioscorea opposita Thunb. by the Quality Center of Guangdong Yifang Pharmaceutical Co., Ltd. It was prepared into bran-fried yam slices according to the Chinese Pharmacopoeia (2020 edition) under the section for bran-fried yam slices. Detailed information is provided in Table 2.

[0208] Table 2 Origin information of 19 batches of Chinese yam medicinal materials and braised Chinese yam slices

[0209]

[0210]

[0211] 1.2 Chromatographic conditions

[0212] A Waters HSS T3 (150 mm × 2.1 mm, 1.8 μm) chromatographic column was used; methanol was used as mobile phase A, 0.03 mol / L ammonium acetate solution was used as mobile phase B, and gradient elution was performed according to the requirements in Table 3 below; the flow rate was 0.3 ml per minute; the column temperature was 30°C; the detection wavelength was 258 nm; and the injection volume was 2 μl.

[0213] Table 3 Gradient elution table

[0214]

[0215] 1.3 Preparation of reference solution

[0216] Take approximately 1 g of Chinese yam control medicinal material and place it in a stoppered conical flask. Add 25 ml of 10% methanol aqueous solution and heat under reflux for 30 minutes. Remove, cool, shake well, filter, and use the filtrate as the control medicinal material reference solution. Accurately weigh 1.039 mg of cytidine, 1.129 mg of adenine, 1.064 mg of uridine, 1.071 mg of guanosine, 1.089 mg of tryptophan, and 1.809 mg of adenosine. Place them in a 25 ml volumetric flask and add 10% methanol aqueous solution to prepare a mixed reference substance stock solution. Accurately pipette 2 ml of the stock solution and place it in a 10 ml volumetric flask. Add 10% methanol aqueous solution to prepare a solution containing 8.262 μg of cytidine, 8.916 μg of adenine, 8.478 μg of uridine, 8.020 μg of guanosine, 8.704 μg of tryptophan, and 14.428 μg of adenosine per 1 ml to obtain the mixed reference substance solution.

[0217] 1.4 Preparation of test solution

[0218] Take about 0.5 g of the sample powder to be tested (passed through a No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 10% methanol aqueous solution, weigh the weight, and ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 10% methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain it.

[0219] 1.5 Methodological Validation

[0220] (1) Precision inspection

[0221] Take an appropriate amount of the sample powder (passed through a No. 4 sieve) and prepare a test solution according to the test solution preparation method specified in "1.4". Inject and analyze according to the chromatographic conditions in "1.2". Inject the same sample solution six times continuously. Using the uridine chromatographic peak as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak to the S peak, and calculate the RSD value. The experimental results are shown in Tables 4 and 5.

[0222] Table 4 Results of precision investigation of characteristic spectrum of Chinese yam medicinal material (relative retention time)

[0223]

[0224] Table 5 Precision investigation results of characteristic spectrum of Chinese yam (relative peak area)

[0225]

[0226] The results showed that when the same sample solution was injected six times continuously, the relative retention time RSD values ​​of each characteristic peak and the S peak were all less than 2%, and the relative peak area RSD values ​​were all less than 5%, indicating that the instrument had good precision.

[0227] (2) Repeatability study

[0228] Take an appropriate amount of the sample powder (passed through a No. 4 sieve) and prepare six test solutions according to the test solution preparation method specified in "1.4". Inject and analyze according to the chromatographic conditions in "1.2". Using the uridine chromatographic peak as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak to the S peak, and calculate the RSD value. The experimental results are shown in Tables 6 and 7.

[0229] Table 6 Repeatability test results of characteristic spectrum of Chinese yam (relative retention time)

[0230]

[0231] Table 7 Repeatability test results of characteristic spectrum of Chinese yam (relative peak area)

[0232]

[0233] The results showed that the same batch of samples were measured 6 times, and the relative retention time RSD values ​​of each characteristic peak and S peak were all less than 2%, and the relative peak area RSD values ​​were all less than 5%, indicating that the method had good repeatability.

[0234] (3) Stability investigation

[0235] Take an appropriate amount of the sample powder (passed through a No. 4 sieve) and prepare a test solution according to the test solution preparation method specified in "1.4". Inject the sample and analyze it at 0, 2, 4, 8, 12, and 24 hours according to the chromatographic conditions in "1.2". Using the uridine chromatographic peak as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak to the S peak, and calculate the RSD value. The experimental results are shown in Tables 8 and 9.

[0236] Table 8 Results of stability study of characteristic spectrum of Chinese yam medicinal material (relative retention time)

[0237]

[0238] Table 9 Results of stability study of characteristic spectrum of Chinese yam medicinal material (relative peak area)

[0239]

[0240] The results showed that when the same sample solution was analyzed at 0, 2, 4, 8, 12, and 24 hours, the relative retention time RSD values ​​of each characteristic peak and the S peak were all less than 2%, and the relative peak area RSD values ​​were all less than 5%, indicating that the sample solution was relatively stable within 24 hours.

[0241] 1.6 Sample determination

[0242] According to the above test sample preparation method and chromatographic conditions, 19 batches of Chinese yam medicinal materials and 19 batches of corresponding batches of bran-fried Chinese yam slices were measured, and their relative retention times and relative peak areas were calculated. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" was used to identify the common peaks of the characteristic spectra of the 19 batches of Chinese yam medicinal materials. The overlay diagram is shown in Figure 1 , and generate a comparison map see Figure 2 ; Yam reference medicinal material atlas see Figure 3 ; Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" to identify the common peaks of the characteristic spectra of 19 batches of corresponding batches of bran fried yam slices, and the overlay diagram is shown in Figure 4 , and generate a comparison map see Figure 5 ; The measurement results are shown in Tables 10 to 13:

[0243] Table 10 Determination results of relative retention time of characteristic spectrum of Chinese yam medicinal material

[0244]

[0245] Table 11 Determination results of relative peak areas of characteristic spectrum of Chinese yam medicinal material

[0246]

[0247] Table 12 Determination results of relative retention time of characteristic spectrum of fried yam slices with bran

[0248]

[0249]

[0250] Table 13 Determination results of relative peak area of ​​characteristic spectrum of fried yam slices with bran

[0251]

[0252]

[0253] 1) The chromatographic peaks of the characteristic spectrum of yam medicinal material were assigned as follows: peak 1 is cytidine; peak 2 is uridine; peak 3 is adenine; peak 4 is guanosine; peak 5 is tryptophan; and peak 6 is adenosine.

[0254] 2) The chromatographic peaks of the characteristic spectrum of the fried yam slices with bran were attributed, which were as follows: peak 2 was cytidine; peak 3 was uridine; peak 5 was adenine; peak 7 was guanosine; peak 9 was tryptophan; and peak 11 was adenosine.

[0255] According to the above test results and Figure 6The comparison chart shows that the characteristic spectrum of the braised yam slices with bran has a chromatographic peak with a high response value (peak 1 and peak 4) before peak 2 (cytidine) and peak 5 (adenine). There are also corresponding high-response peaks (peak 6, peak 8, peak 10) on both sides of peak 7 (guanosine) and between peak 9 (tryptophan) and peak 11 (adenosine), which are significantly different from the characteristic spectrum of yam medicinal material. The two can be distinguished based on the characteristic peaks of the characteristic spectrum.

[0256] 1.7 Counterfeit Comparison

[0257] According to the above test sample preparation method and chromatographic conditions, 2 batches of ginseng, 3 batches of Chinese yam, and 3 batches of cassava were tested. The results are shown in the figure. Figure 7 , calculate the relative retention time and relative peak area, the results are shown in Table 14 to Table 15:

[0258] Table 14 Relative retention time determination results of characteristic spectra of three counterfeit yam products

[0259]

[0260]

[0261] Table 15 Comparison of relative peak areas of characteristic spectra of Chinese yam and three counterfeit Chinese yam

[0262]

[0263]

[0264] The above results show that Peak 6 (adenosine) was not detected in the ginseng potato characteristic profile, and Peak 3 (adenine) had a high response value and a larger relative peak area than yam. Peak 1 (cytidine) was not detected in one of the three batches of yam, while the relative peak areas of Peak 1 in the remaining two batches were very small, and Peak 6 had a smaller relative peak area than yam. Peak 3 (adenine) had a high response value in cassava and a larger relative peak area than yam. Therefore, the relative peak area specification can be used to distinguish yam from its counterfeits.

[0265] 1.8 Feature Map Preparation

[0266] Based on the above results, the characteristic spectrum standard of yam medicinal material was finally determined as follows: 6 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the control medicinal material reference; 5 of the characteristic peaks are consistent with the retention time of the reference, and the peak corresponding to the uridine reference is the S peak. The relative retention time of characteristic peak 1 and S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: 0.65 (peak 1); the relative peak areas of peak 3, peak 6 and S peak are calculated, and the specified values ​​are: peak 1 shall not be greater than 0.26, and peak 6 shall not be less than 1.3.

[0267] The final characteristic spectrum standard of fried yam slices with bran was determined as follows: 11 characteristic peaks should be present in the chromatogram of the test sample, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the control medicinal material reference; 5 of the characteristic peaks are consistent with the retention times of the reference, the peak corresponding to the uridine reference is the S1 peak, and the relative retention times of characteristic peaks 1, 2, and 4 to the S1 peak are calculated, and the relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.52 (peak 1), 0.65 (peak 3), and 1.39 (peak 4); the peak corresponding to the guanosine reference is the S2 peak, and the relative retention times of characteristic peaks 6, 8, and 10 to the S2 peak are calculated, and the relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.97 (peak 5), 1.07 (peak 7), and 1.47 (peak 9).

[0268] Comparative Example 1

[0269] (1) Preparation of reference solution:

[0270] Accurately weigh 3.475 mg of adenosine reference substance, place it in a 25 ml volumetric flask, add 10% methanol aqueous solution to make a mother solution containing 138.583 μg per 1 ml, accurately pipette 1 ml of the mother solution, place it in a 25 ml volumetric flask, add 10% methanol aqueous solution to make a solution containing 5.543 μg per 1 ml.

[0271] (2) Preparation of test solution:

[0272] Take about 0.5 g of the powder to be tested (passed through a No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of a 10% methanol aqueous solution, weigh it, and ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with a 10% methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain it.

[0273] (3) Chromatographic conditions:

[0274] The chromatographic column was Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm); the mobile phase was methanol-water (7:93); the flow rate was 1.0 ml / min; the column temperature was 30 °C; the detection wavelength was 258 nm. The chromatographic results are shown in Figure 8 .

[0275] Depend on Figure 8 It can be seen that when methanol-water is used as the mobile phase for isocratic elution, the adenosine chromatographic peak emerges quickly and fails to achieve the effect of separating and identifying multiple compounds, so the purpose of differential analysis cannot be achieved.

[0276] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0277] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of yam, characterized in that: The steps include: Take a yam sample to be tested and prepare a test solution; the yam sample to be tested is selected from yam medicinal materials or bran-fried yam slices; The test solution is subjected to ultra-high performance liquid chromatography analysis to construct a characteristic spectrum of yam; Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include: Gradient elution with a mobile phase was used, wherein the mobile phase included a mobile phase A and a mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution; the concentration of ammonium acetate in the aqueous ammonium acetate solution was 0.02 mol / L to 0.03 mol / L; The procedure of the gradient elution is: From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%; From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%; From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%; From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%; From 40 to 45 minutes, the volume percentage of mobile phase A was changed from 30% to 65%, and the volume percentage of mobile phase B was changed from 70% to 35%; The chromatographic column was a Waters HSS T3 column, 150 mm × 2.1 mm, 1.8 μm; The wavelength is 250nm~270nm; The preparation of the test solution includes the following steps: Mixing the yam sample to be tested with an alcohol-containing aqueous solution, performing ultrasonic extraction, filtering, and collecting a filtrate; The method further comprises the steps of preparing a mixed reference substance solution and performing ultra-high performance liquid chromatography analysis on the mixed reference substance solution; The preparation of the mixed reference substance solution includes the following steps: Cytidine, uridine, adenine, guanosine, tryptophan, adenosine and a methanol aqueous solution with a methanol volume percentage of 5% to 20% are mixed.

2. The method for constructing a characteristic spectrum of Chinese yam according to claim 1, wherein: The chromatographic conditions of the ultra-high performance liquid chromatography analysis also include: a column temperature of 28° C. to 32° C.; and a flow rate of 0.28 mL / min to 0.32 mL / min.

3. The method for constructing a characteristic spectrum of Chinese yam according to any one of claims 1 to 2, characterized in that: The method also includes the steps of preparing a control medicinal material reference solution and performing ultra-high performance liquid chromatography analysis on the control medicinal material reference solution.

4. The method for constructing a characteristic spectrum of Chinese yam according to claim 3, wherein: The solvents in the control medicinal material reference solution and the mixed reference substance reference solution are independently selected from one or a mixture of an alcohol-containing aqueous solution and water.

5. The method for constructing a characteristic spectrum of Chinese yam according to claim 4, wherein: The preparation of the control medicinal material reference solution comprises the following steps: The Chinese yam control medicinal material was mixed with the alcohol-containing aqueous solution, heated to reflux, filtered, and the filtrate was collected.

6. A method for detecting yam, characterized in that: The steps include: Taking a yam sample to be tested and preparing a yam sample solution to be tested; Performing ultra-high performance liquid chromatography analysis on the yam sample solution to be tested, and comparing the obtained chromatogram with the characteristic spectrum of yam constructed by the method for constructing the characteristic spectrum of yam according to any one of claims 1 to 5; Among them, the chromatographic conditions of ultra-high performance liquid chromatography analysis include: Gradient elution with a mobile phase was used, wherein the mobile phase included a mobile phase A and a mobile phase B, wherein the mobile phase A was methanol and the mobile phase B was an aqueous ammonium acetate solution; the concentration of ammonium acetate in the aqueous ammonium acetate solution was 0.02 mol / L to 0.03 mol / L; The procedure of the gradient elution is: From 0 min to 10 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%; From 10 min to 22 min, the volume percentage of mobile phase A was changed from 0% to 3%, and the volume percentage of mobile phase B was changed from 100% to 97%; From 22 to 30 minutes, the volume percentage of mobile phase A changed from 3% to 12%, and the volume percentage of mobile phase B changed from 97% to 88%; From 30 to 40 minutes, the volume percentage of mobile phase A was changed from 12% to 30%, and the volume percentage of mobile phase B was changed from 88% to 70%; From 40 to 45 minutes, the volume percentage of mobile phase A was changed from 30% to 65%, and the volume percentage of mobile phase B was changed from 70% to 35%; The chromatographic column was a Waters HSS T3 column, 150 mm × 2.1 mm, 1.8 μm; The wavelength is 250nm~270nm; The preparation of the yam sample solution to be tested comprises the following steps: The yam sample to be tested is mixed with an alcohol-containing aqueous solution, subjected to ultrasonic extraction, filtered, and the filtrate is collected.

7. The method for detecting Chinese yam according to claim 6, wherein: The chromatographic conditions of the ultra-high performance liquid chromatography analysis also include: a column temperature of 28° C. to 32° C.; and a flow rate of 0.28 mL / min to 0.32 mL / min.

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