Characteristic chromatogram construction method for distinguishing dioscorea opposita thunb and adulterants thereof based on HPLC-CAD and application of characteristic chromatogram construction method

Abstract

The invention relates to a specific chromatogram construction method for distinguishing iron stick yam and counterfeit products thereof based on HPLC-CAD (High Performance Liquid Chromatography-Computer Aided Design) and application of the specific chromatogram construction method, the iron stick yam and the counterfeit products thereof can be effectively distinguished, and the quality and the medicinal value of the iron stick yam are guaranteed. Then, detecting by using a high performance liquid chromatography electrospray detector under specific chromatographic conditions, and finally, according to a detection result, drawing a standard characteristic chromatogram of the iron stick yam medicinal material as well as the counterfeit product and the reference medicinal material of the iron stick yam medicinal material; the specific chromatogram is applied to quality detection, evaluation and control of the Tiegun yam medicinal material, or is applied to distinguishing the Tiegun yam and counterfeit products of the Tiegun yam. According to the specific chromatogram construction method, the iron stick yam and the counterfeit potatoes, cassava, sweet potatoes and dioscorea nipponica can be identified according to whether the raffinose exists in the chromatographic peak or not, the product quality and medicinal value of the iron stick yam are effectively guaranteed, and the specific chromatogram construction method has practical application value.

Description

Technical Field

[0001] This invention relates to Chinese medicinal materials, and in particular to a method for constructing characteristic chromatograms based on HPLC-CAD to distinguish between Dioscorea opposita and its adulterants, and its application. Background Technology

[0002] Yam is a species included in Part I of the Chinese Pharmacopoeia (2025 edition), and its source is the plant Dioscorea opposita (Dioscorea opposita) of the Dioscoreaceae family. Dioscorea oposita The dried rhizome of *Thunb.* is neutral in nature and sweet in taste, possessing the effects of tonifying the spleen and stomach, promoting body fluid production and benefiting the lungs, and tonifying the kidneys and astringing essence. It is a model variety of food and medicine sharing the same origin. Modern research shows that yam contains abundant polysaccharides, saponins, polyphenols, amino acids, and other functional components, possessing antioxidant, hypoglycemic, hypolipidemic, and gastrointestinal regulatory activities; oligosaccharides, in particular, are its main functional active ingredients, and have been developed into multifunctional health products abroad. Henan is one of the authentic producing areas of yam, called "Huai yam," especially the iron rod yam, named for its rust-like surface, fine and sparse root hairs, hard texture, and iron rod shape. It has excellent qualities such as "white flesh, sweet taste, and firm texture," and has high medicinal and edible value. However, with the increasing market demand for iron rod yam, many closely related varieties such as cassava (Euphorbia milii) are also being sold. Manihot esculenta Crantz), Convolvulaceae sweet potato ( Ipomoea batatas Lam.), Dioscorea opposita (Dioscorea opposita), a plant in the Dioscoreaceae family. Dioscorea alata Using non-yam varieties such as L. (often called *Dioscorea opposita*) in medicine raises concerns about clinical efficacy and safety. There is an urgent need for efficient and accurate identification of *Dioscorea opposita* and its closely related hybrids to support the development and utilization of *Dioscorea opposita* germplasm resources and the stable development of the market.

[0003] Currently, there are reports on the identification of *Dioscorea opposita* and its adulterants using DNA barcoding, microscopic features, near-infrared spectroscopy, and HPLC characteristic chromatograms. However, there are no reports on the identification of carbohydrate components in *Dioscorea opposita* using HPLC-CAD characteristic chromatograms. Electrospray ionization (CAD) is a novel detector developed in recent years. Compared with ultraviolet detectors, refractive index detectors (RID), and evaporative light scattering detectors (ELSD), it has higher sensitivity, better stability, reproducibility, and robustness, and is effective in determining monosaccharides and oligosaccharides. Therefore, developing a method for constructing characteristic chromatograms based on HPLC-CAD to distinguish *Dioscorea opposita* and its adulterants for the identification of genuine *Dioscorea opposita* is a technical problem that needs to be solved. Summary of the Invention

[0004] In view of the above situation, the purpose of this invention is to provide a method for constructing characteristic chromatograms based on HPLC-CAD to distinguish iron yam and its adulterants, and its application, which can effectively distinguish iron yam from its adulterants and ensure the quality and medicinal value of iron yam.

[0005] The technical solution provided by this invention is a method for constructing characteristic chromatograms to distinguish between Dioscorea opposita and its adulterants based on HPLC-CAD, which is achieved through the following steps: S1. Preparation of the test solution of Dioscorea opposita: Weigh an appropriate amount of genuine Dioscorea opposita, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, and add water in a volume equal to 100 times the weight of the medicinal material each time. The weight and volume refer to g for solids and mL for liquids (the same below). Heat and reflux to extract 1-2 times, each extraction time being 1-2 hours. Combine the extracts, cool to room temperature, concentrate under reduced pressure to 30% of the added water volume, then add anhydrous ethanol to make the alcohol content of the solution reach 85%-95% by volume. Shake well, let stand at 4℃ for 12 hours, and discard the supernatant. Wash the precipitate with anhydrous ethanol and acetone in sequence. After washing, centrifuge at 4000r for 10 minutes, discard the supernatant, obtain the precipitate, evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight, obtain powder, dissolve in distilled water to obtain the test solution of Dioscorea opposita. S2. Preparation of the adulterated medicinal material solution: Weigh an appropriate amount of adulterated medicinal material, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, each time adding water at a volume 100 times the weight of the medicinal material, heat and reflux to extract 1-2 times, each extraction time is 1-2 hours, combine the extracts, cool to room temperature, concentrate under reduced pressure to 30% of the added water volume, then add anhydrous ethanol to make the solution contain 85%~95% alcohol by volume, shake well, let stand at 4℃ for 12 hours, discard the supernatant; wash the precipitate with anhydrous ethanol and acetone in sequence, after washing, centrifuge at 4000r for 10min, discard the supernatant to obtain the precipitate, then evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight to obtain powder, add distilled water to dissolve, to obtain the adulterated medicinal material solution; The adulterated medicinal material is selected from Dioscorea opposita, a plant of the Dioscoreaceae family and the Dioscorea genus. Dioscorea persimilis Prain & Burkill) and ginseng ( Dioscorea alata L.), cassava (a plant belonging to the genus Cassava of the family Euphorbiaceae) Manihot esculenta Crantz) and sweet potato (Ipomoea spp., a plant in the Convolvulaceae family) Ipomoea batatas (L.) Lam.); S3. Preparation of the reference solution: Weigh 8-22 mg of D-fructose, 13-20 mg of D-glucose, 20-20 mg of sucrose, 8-19 mg of fructosyl sucrose, and 5-18 mg of raffinose, dissolve in distilled water to prepare a solution with a mass concentration of 1-10 mg / mL. -1 The solution is then used to obtain a mixed reference solution; S4, detecting the test product solution, the pseudo-product medicinal material solution and the control product solution by a high performance liquid chromatography-electrospray detector, and the chromatographic conditions are as follows: an Asahipak NH2P-504E chromatographic column (4.6 mm x 250 mm, 3.5 μm) is used, the column temperature is 40 DEG C, gradient elution is carried out at a flow rate of 1 mL / min, and acetonitrile (A) and water (B) are used as mobile phases; the gradient elution program is as follows: 0-8 min, 75% A; 8-10 min, 75% A-71% A; 10-15 min, 71% A; 15-20 min, 71-69% A; 20-25 min, 69% A; a Cornoa VEO SD electrospray detector (a commercially available product) is used for detection, and the parameter settings are as follows: acquisition frequency 50 Hz, filter constant 2.0, air pressure 55.0 Pa; according to the detection results, a standard characteristic spectrum of the D. esculenta var. sinica medicinal material and its pseudo-products and control medicinal materials is drawn; The standard characteristic spectrum of the D. esculenta var. sinica includes five characteristic peaks, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, peak 4 is raffinose and peak 5 is raffinose. The standard characteristic spectrum of the pseudo-product includes four characteristic peaks, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose and peak 4 is raffinose.

[0006] The characteristic spectrum constructed by the above method is applied to quality detection, evaluation and control of the D. esculenta var. sinica medicinal material, or is applied to distinguishing the D. esculenta var. sinica and its pseudo-products.

[0007] The characteristic spectrum construction method for distinguishing the D. esculenta var. sinica and its pseudo-products based on HPLC-CAD determines five components: D-fructose, D-glucose, sucrose, raffinose and raffinose, constructs a characteristic spectrum, and is applied to quality detection, evaluation and control of the D. esculenta var. sinica medicinal material, or is applied to distinguishing the D. esculenta var. sinica and pseudo-products, is simple in operation, short in time consumption, strong in characteristic and good in reproducibility, can identify the D. esculenta var. sinica and its pseudo-products Dioscorea alishanensis, Manihot esculenta, Ipomoea batatas and D. fordii according to the presence or absence of the chromatographic peak raffinose, effectively guarantees product quality and medicinal effect of the D. esculenta var. sinica, and has practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 The chromatogram is a mixed control product solution, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, peak 4 is raffinose and peak 5 is raffinose; Figure 2 The HPLC-CAD chromatogram of the D. esculenta var. sinica sample is shown in the figure, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, peak 4 is raffinose and peak 5 is raffinose; Figure 3HPLC-CAD chromatogram of Dioscorea panthaica sample, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is sucrose fructose trisaccharide. Figure 4 HPLC-CAD chromatogram of Dioscorea panthaica sample, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is sucrose fructose trisaccharide. Figure 5 HPLC-CAD chromatogram of Dioscorea panthaica sample, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is sucrose fructose trisaccharide. Figure 6 HPLC-CAD chromatogram of Dioscorea panthaica sample, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is sucrose fructose trisaccharide. DETAILED DESCRIPTION

[0009] The specific embodiments of the present application are described in detail below in combination with examples and specific cases.

[0010] The present application is given in the following embodiments in the specific implementation: EMBODIMENT

[0011] The present application is given in the following embodiments in the specific implementation: S1, preparation of Dioscorea ferocissima test solution: 1 g of true Dioscorea ferocissima was weighed, dried and crushed, sieved through a 60-mesh sieve, placed in a round-bottom flask, 100 mL of pure water was added, heated and refluxed for 2 times, each time for 2 h, the extract was combined, cooled to room temperature, reduced to 30 mL under reduced pressure, then anhydrous ethanol was added to make the solution contain 85% alcohol by volume, shaken, and placed at 4°C for 12 h, the supernatant was discarded; the precipitate was washed with anhydrous ethanol and acetone in turn, after washing, centrifuged at 4000 r for 10 min, the supernatant was discarded, the precipitate was obtained, and then dried to remove alcohol, vacuum freeze-dried to constant weight to obtain a powder, which was dissolved in distilled water to obtain the Dioscorea ferocissima test solution; S2, preparation of pseudo medicinal material solution: 1 g of pseudo medicinal material was weighed, dried and crushed, sieved through a 60-mesh sieve, placed in a round-bottom flask, 100 mL of pure water was added each time, heated and refluxed for 2 times, each time for 2 h, the extract was combined, cooled to room temperature, reduced to 30 mL under reduced pressure, then anhydrous ethanol was added to make the solution contain 85% alcohol by volume, shaken, and placed at 4°C for 12 h, the supernatant was discarded; the precipitate was washed with anhydrous ethanol and acetone in turn, after washing, centrifuged at 4000 r for 10 min, the supernatant was discarded, the precipitate was obtained, and then dried to remove alcohol, vacuum freeze-dried to constant weight to obtain a powder, which was dissolved in distilled water to obtain the pseudo medicinal material solution; The pseudo control medicinal material is selected from Dioscoreaceae Dioscorea plants such as Dioscorea panthaica (Dioscorea persimilis Prain & Burkill) and Dioscorea alata (Lour. Dioscorea alata Crantz) and Ipomoea batatas (L.) Lam. Manihot esculenta Crantz) and Ipomoea batatas (L.) Lam. Ipomoea batatas Crantz) and Ipomoea batatas (L.) Lam. S3, preparation of a control solution: 21.40 mg of D-fructose, 19.45 mg of D-glucose, 19.55 mg of sucrose, 15.90 mg of raffinose, and 17.55 mg of raffinose were weighed and dissolved in distilled water to prepare a solution with a mass concentration of 4.28, 3.89, 3.91, 3.18, and 3.51 mg / mL, respectively, to obtain a mixed control solution; -1 S3, preparation of a control solution: 21.40 mg of D-fructose, 19.45 mg of D-glucose, 19.55 mg of sucrose, 15.90 mg of raffinose, and 17.55 mg of raffinose were weighed and dissolved in distilled water to prepare a solution with a mass concentration of 4.28, 3.89, 3.91, 3.18, and 3.51 mg / mL, respectively, to obtain a mixed control solution; S4, detecting the test solution, the pseudo-drug material solution, and the control solution by HPLC-ESI, and the chromatographic conditions are as follows: using an Asahipak NH2P-504E chromatographic column (4.6 mm x 250 mm, 3.5 μm), column temperature 40℃, gradient elution at a flow rate of 1 mL / min, using acetonitrile (A) and water (B) as the mobile phase; the gradient elution program is as follows: 0~8 min, 75% A; 8~10 min, 75% A~71% A; 10~15 min, 71% A; 15~20 min, 71~69% A; 20~25 min, 69% A; using a Cornoa VEO SD ESI detector (commercial product), and the parameters are set as follows: acquisition frequency 50Hz, filter constant 2.0, gas pressure 55.0 Pa; according to the detection results, the standard characteristic spectrum of the control drug material and the pseudo-drug material of the iron stick yam is drawn; The standard characteristic spectrum of the iron stick yam includes five characteristic peaks, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, peak 4 is raffinose, and peak 5 is raffinose; The standard characteristic spectrum of the pseudo-drug material includes four characteristic peaks, peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is raffinose. Example

[0012] The characteristic spectrum construction method for distinguishing the iron stick yam and its pseudo-drugs based on HPLC-CAD is realized by the following steps: S1, preparation of the test solution of the true product of the iron stick yam: 2g of the true product of the iron stick yam is weighed, dried and crushed, passed through a 60-mesh sieve, placed in a round-bottom flask, 200ml of pure water is added each time, heated to reflux extraction for 2h, the extraction is carried out twice, the extraction liquid is cooled to room temperature, concentrated to 60ml under reduced pressure, then anhydrous ethanol is added to make the alcohol content of the solution reach 95%, shake well, stand at 4 DEG C for 12h, discard the supernatant; the precipitate is washed with anhydrous ethanol and acetone in turn, after washing, centrifugation at 4000r for 10min, the supernatant is discarded, the precipitate is obtained, and then dried to no alcohol smell, vacuum freeze-dried to constant weight, powder is obtained, dissolved in distilled water to obtain the test solution of the iron stick yam; S2, preparation of the solution of the pseudo medicinal material: 2g of the pseudo medicinal material is weighed, dried and crushed, passed through a 60-mesh sieve, placed in a round-bottom flask, 200ml of pure water is added each time, heated to reflux extraction for 2h, the extraction is carried out twice, the extraction liquid is cooled to room temperature, the extraction liquid is combined, cooled to room temperature, concentrated to 60ml under reduced pressure, then anhydrous ethanol is added to make the alcohol content of the solution reach 95%, shake well, stand at 4 DEG C for 12h, discard the supernatant; the precipitate is washed with anhydrous ethanol and acetone in turn, after washing, centrifugation at 4000r for 10min, the supernatant is discarded, the precipitate is obtained, and then dried to no alcohol smell, vacuum freeze-dried to constant weight, powder is obtained, dissolved in distilled water to obtain the solution of the pseudo medicinal material; S3, preparation of the control solution: 12.56mg of D-fructose, 15.78mg of D-glucose, 15.82mg of sucrose, 18.36mg of raffinose and 17.02mg of stachyose are weighed, dissolved in distilled water to prepare a solution with a mass concentration of 6.28, 7.89, 7.91, 9.18, 8.51 mg·mL -1 of the mixed control solution is obtained; S4, the test solution, the solution of the pseudo medicinal material and the control solution are detected according to the high performance liquid chromatography electrospray detector, and the standard characteristic spectrum of the iron stick yam control medicinal material and the pseudo medicinal material control medicinal material is drawn according to the detection result. Embodiment

[0013] The characteristic spectrum construction method for distinguishing the iron stick yam and the pseudo medicinal material thereof based on HPLC-CAD of the application is realized by the following steps: S1, preparation of the test solution of the true product of the iron stick yam: 5g of the true product of the iron stick yam is weighed, dried and crushed, passed through a 60-mesh sieve, placed in a round-bottom flask, and then 500ml of pure water is added each time, heated to reflux for 2h, extracted twice, and the filtrate is combined; the extract is cooled to room temperature, and then 150ml of distilled water is added, followed by the addition of anhydrous ethanol to make the solution contain 90% alcohol by volume, shaken well, and then placed at 4 DEG C for 12h, and the supernatant is discarded; the precipitate is washed with anhydrous ethanol and acetone in turn, and then centrifuged at 4000r for 10min after washing, and the supernatant is discarded, and the precipitate is dried to remove the alcohol, and then vacuum freeze-dried to a constant weight to obtain a powder, which is dissolved in distilled water to obtain the test solution of the true product of the iron stick yam; S2, preparation of the solution of the pseudo-product medicinal material: 5g of the pseudo-product medicinal material is weighed, dried and crushed, passed through a 60-mesh sieve, placed in a round-bottom flask, and then 500ml of pure water is added each time, heated to reflux for 2h, extracted twice, and the filtrate is combined; the extract is cooled to room temperature, and then 150ml of distilled water is added, followed by the addition of anhydrous ethanol to make the solution contain 90% alcohol by volume, shaken well, and then placed at 4 DEG C for 12h, and the supernatant is discarded; the precipitate is washed with anhydrous ethanol and acetone in turn, and then centrifuged at 4000r for 10min after washing, and the supernatant is discarded, and the precipitate is dried to remove the alcohol, and then vacuum freeze-dried to a constant weight to obtain a powder, which is dissolved in distilled water to obtain the solution of the pseudo-product medicinal material; S3, preparation of the control solution: 8.56mg of D-fructose, 13.78mg of D-glucose, 15.82mg of sucrose, 8.36mg of raffinose, and 5.02mg of raffinose are weighed, dissolved in distilled water to prepare a solution with a mass concentration of 4.28, 6.89, 7.91, 4.18, and 2.51mg·mL -1 respectively, and a mixed control solution is obtained; S4, the test solution, the solution of the pseudo-product medicinal material and the control solution are detected by high performance liquid chromatography-electrospray detector, and according to the detection results, the standard characteristic spectrum of the true product of the iron stick yam and the pseudo-product of the iron stick yam is drawn.

[0014] The characteristic spectrum constructed by the above method is applied in the quality detection, evaluation and control of the iron stick yam medicinal material, or is applied in the differentiation of the iron stick yam and the pseudo-product thereof.

[0015] The characteristic spectrum construction method for distinguishing the iron stick yam and the pseudo-product thereof based on HPLC-CAD in the application determines five components: D-fructose, D-glucose, sucrose, raffinose and raffinose, constructs a characteristic spectrum, and is applied in the quality detection, evaluation and control of the iron stick yam medicinal material, or is applied in the differentiation of the iron stick yam and the pseudo-product, and very good beneficial technical effects are obtained, and the test data are as follows: In order to establish a reasonable HPLC-CAD identification method, and improve the extraction efficiency of monosaccharides and oligosaccharides of iron stick yam and its counterfeit, the present application is investigated from the following aspects: ① Selection of extraction solvent: different concentrations of pure water, methanol and ethanol are selected as solvents in the experiment, and the influence of the above solvents on the content of the measured components is investigated. The results show that when pure water is selected as the solvent, the content of monosaccharides and oligosaccharides is the highest, and the extraction is the most sufficient, so pure water is selected as the solvent in the experiment; ② Selection of extraction time and times: in the preparation of the test solution, the results show that when the heating reflux time is 1.5 h and the extraction times is 2 times, the sugar yield is the highest; ③ Selection of different chromatographic columns: Waters XBridge HILIC (4.6 mm x 150 mm, 3.5 um) and Asahipak NH2P-504E (4.6 mm x 250 mm, 3.5 um) chromatographic columns are investigated, and the results show that the Asahipak NH2P-504E4 chromatographic column has a stable baseline, good chromatogram peak type and separation degree; ④ Selection of mobile phase: acetonitrile-water, methanol-water, acetonitrile-0.1% formic acid and methanol-0.1% formic acid are investigated as the mobile phase for gradient elution, and the results show that acetonitrile is selected as the mobile phase A and pure water is selected as the mobile phase B, and the HPLC-CAD chromatogram has good peak type and separation degree; ⑤ Selection of column temperature: 30℃, 40℃ and 50℃ column temperatures are investigated, and the results show that when the column temperature is 40℃, the chromatographic peak fluctuation is smaller. According to the above, the HPLC-CAD method for distinguishing iron stick yam and its close relative counterfeit is finally determined.

[0016] The purpose, technical solution and the like of the present application are described in detail in combination with specific embodiments, but do not constitute any limitation on the present application, and any limited modification of the present application still belongs to the scope of the present application.

[0017] Experiment one: The method of the present application is used for HPLC-CAD detection of iron stick yam from different producing areas, and the specific steps are as follows: 1. Experimental materials 1.1. Experimental instruments CoronaTM VE0TM electrospray detector (Thermo Fisher Scientific Corporation); Asahipak NH2P-504E chromatographic column (4.6 mm x 250 mm, 3.5 um) 1.2. Reagents and medicinal materials The control samples were purchased from Chengdu Zhizhuanghua Pure Biotechnology Co., Ltd., with a purity of 99.9%; Fisher chromatography methanol and chromatography acetonitrile were purchased from Thermo Fisher Scientific, and distilled water was Wahaha pure water.

[0018] Iron stick yam samples were used in November 2024 in Henan Wenxian, Shandong Dingtao, Shanxi Yuncheng, Hebei Lixian, a total of 30 batches of medicinal materials, 15 batches of iron stick yam in Henan Wenxian, 5 batches of iron stick yam in Shandong, Shanxi and Hebei (Table 1), for HPLC-CAD detection.

[0019] Table 1 Iron stick yam sample information

[0020] 2、Experimental method 2.1、Preparation of test solution Precisely take 1 g of iron stick yam powder (pass through No. 4 sieve) and put it in a 250 mL round-bottom flask, add 100 mL of water, heat reflux for 1.5 h, filter while hot; repeat the above extraction once for the residue, and combine the two filtrates. Cool the filtrate, concentrate under reduced pressure to 30 mL, then add anhydrous ethanol to make the alcohol content of the solution reach 90%, shake well, and stand at 4°C for 12 h. Discard the supernatant, and the precipitate is washed with anhydrous ethanol twice and acetone once, each time after washing, centrifuge at 4000 r / min for 10 min and discard the supernatant. Dry the precipitate to no alcohol smell, then vacuum freeze-dry to constant weight; take 50 mg of freeze-dried powder, accurately add 1 mL of distilled water, dissolve, and obtain the test solution of iron stick yam monosaccharides and oligosaccharides.

[0021] 2.2、Preparation of mixed control solution Weigh 21.40 mg of D-fructose, 19.45 mg of D-glucose, 19.55 mg of sucrose, 15.90 mg of raffinose, and 17.55 mg of raffinose, dissolve in 5 mL of distilled water, to prepare a mixed solution with a mass concentration of 4.28, 3.89, 3.91, 3.18, and 3.51 mg·mL -1 , respectively, namely the mixed control solution (Example 1).

[0022] 2.3、Chromatographic conditions The Asahipak NH2P-504E column (4.6 mm x 250 mm, 3.5 μm) was used with a column temperature of 40 ℃ and a flow rate of 1 mL / min. The mobile phase was acetonitrile (A) and water (B) with the following gradient elution program: 0-8 min, 75% A; 8-10 min, 75% A to 71% A; 10-15 min, 71% A; 15-20 min, 71-69% A; 20-25 min, 69% A. The Cornoa VEO SD electrospray detector was used with the following parameters: acquisition frequency 50 Hz, filter constant 2.0, and gas pressure 55.0 Pa. The method can achieve good separation of the chromatographic peaks.

[0023] 2.4. Methodology investigation (1) Linear relationship investigation 0.5, 1.0, 2.5, 5.0, 10.0 mL of the mixed reference substance under item “2.2” were taken into 10 mL volumetric flasks, distilled water was added to dilute to the calibration mark, and shaken well. 10 μL of each of the series of concentration reference substance solutions was taken, and chromatographic analysis was performed under the chromatographic conditions of item “2.3”. The corresponding reference substance concentration (x) was taken as the abscissa, and the corresponding peak area (y) was taken as the ordinate, to draw a standard curve, and the results are shown in Table 2. X Y

[0024] Table 2. Linear relationship investigation results

[0025] (2) Precision test The test sample solution (HN1) prepared under item “2.1” was precisely taken, and 6 injections were continuously performed under the chromatographic conditions of item “2.3”. The RSDs of the peak areas of D-fructose, D-glucose, sucrose, raffinose, and raffinose were 1.76%, 1.25%, 1.03%, 0.98%, and 1.4%, respectively, indicating that the precision of the determination was good.

[0026] (3) Reproducibility test The same iron stick yam sample (HN1) was precisely weighed, and 6 aliquots were prepared according to the method of item “2.1”. 10 μL of each test sample solution was injected into the liquid chromatograph, and the determination was performed according to the chromatographic conditions of item “2.4.3”. The average contents of D-fructose, D-glucose, sucrose, raffinose, and raffinose were 5.60, 2.24, 3.61, 2.51, and 1.13 mg·g -1 , respectively, and the RSDs were 1.94%, 1.71%, 1.65%, 1.26%, and 1.70%, respectively, indicating that the reproducibility of the method was good. ​​

[0027] (4) Stability test Take the test solution (HN1) under item "2.1", after precise suction, sample determination at 0, 2, 4, 8, 12, 24 h. The results show that the RSD of D-fructose, D-glucose, sucrose, raffinose, and stachyose peak area is 0.67%, 0.59%, 0.87%, 1.24%, and 1.40%, respectively, indicating that the test solution has good stability within 24 h.

[0028] (5) Sample recovery rate test Precisely weigh 0.5 g of iron stick yam powder (HN1) with known content, and add 0.5 g of control product at a ratio of 1:1 of sample content. Prepare the test solution according to the method under item "2.1", operate in parallel for 6 times, inject 10 μL, and determine according to the chromatographic conditions under item "2.3", and calculate the sample recovery rate. The results are shown in Table 3, indicating that the method has good recovery rate.

[0029] Table 3 Recovery determination results of 5 monosaccharide / oligosaccharide components in iron stick yam

[0030] 3. Results Precisely take 10 μL of iron stick yam test solution and inject it into HPLC-CAD chromatograph, and determine the absorbance of each sample according to the method under item "2.3". The HPLC-CAD chromatographic peaks of iron stick yam medicinal materials are attributed as follows: peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, peak 4 is raffinose, and peak 5 is stachyose. According to the calculation results of the standard curve, the content of monosaccharide and oligosaccharide in different iron stick yam samples is calculated as dry product. The results are shown in Table 4. Figure 2

[0031] Table 4 Determination results of 5 components in iron stick yam (n=3, %)

[0032] Experiment two ​The HPLC-CAD method of this invention was used to detect the chromatographic peaks and contents of monosaccharides and oligosaccharides in *Dioscorea opposita* samples (6 batches, sample numbers Dp1 ~ Dp6) from Guangdong and Guangxi Zhuang Autonomous Regions. 1 g of powdered *Dioscorea opposita* (passed through a No. 4 sieve) from each sample was placed in a 250 mL round-bottom flask, 100 mL of water was added, and the mixture was heated under reflux for 1.5 h. The mixture was then filtered while hot. The residue was extracted once more, and the two filtrates were combined. The filtrate was cooled and concentrated under reduced pressure to approximately 30 mL. Anhydrous ethanol was then added to bring the alcohol content to 90%, and the solution was shaken well and allowed to stand at 4 °C for 12 h. The supernatant was discarded, and the precipitate was washed twice with anhydrous ethanol and once with acetone. After each washing, the precipitate was centrifuged at 4000 r / min for 10 min, and the supernatant was discarded. After evaporating the precipitate until no alcohol odor remains, freeze-dry it under vacuum to constant weight. Take 50 mg of the freeze-dried powder and accurately add 1 mL of distilled water. After dissolving, the monosaccharide and oligosaccharide test solutions of *Dioscorea opposita* are obtained. Accurately inject 10 μL of the *Dioscorea opposita* test solution into the HPLC-CAD chromatograph and determine the absorbance of each sample according to the method in section "2.3". The HPLC-CAD chromatographic peaks of *Dioscorea opposita* (…) Figure 3 The peaks were assigned as follows: peak 1 was D-fructose, peak 2 was D-glucose, peak 3 was sucrose, and peak 4 was fructosaccharide. No raffinose peak was detected. Based on the results of the standard curve, the content of monosaccharides and oligosaccharides in the dried yam samples was calculated, and the results are shown in Table 5.

[0033] Table 5. Results of determination of four sugar components in Dioscorea opposita (n=3, %)

[0034] Experiment 3 Using the method of this invention, HPLC-CAD analysis was performed on ginseng and yam samples from different origins (a total of 6 batches, sample numbers Da1 to Da6). 1 g of powdered ginseng and yam (passed through a No. 4 sieve) from each sample was placed in a 250 mL round-bottom flask, 100 mL of water was added, and the mixture was heated under reflux for 1.5 h. The mixture was then filtered while hot. The residue was extracted once more, and the two filtrates were combined. The filtrate was cooled and concentrated under reduced pressure to approximately 30 mL. Anhydrous ethanol was then added to bring the alcohol content to 90%, and the mixture was shaken well and allowed to stand at 4°C for 12 h. The supernatant was discarded, and the precipitate was washed twice with anhydrous ethanol and once with acetone. After each washing, the precipitate was centrifuged at 4000 r / min for 10 min, and the supernatant was discarded. The precipitate was evaporated until no alcohol odor remained, and then freeze-dried under vacuum to constant weight. 50 mg of the freeze-dried powder was precisely added to 1 mL of distilled water, and after dissolution, the ginseng and yam monosaccharide and oligosaccharide test solutions were obtained.

[0035] The precise aspirate sample solution of the Dioscorea alata is injected into the HPLC-CAD chromatograph, and the absorbance of each sample is determined according to the method in item 2.3. The HPLC-CAD chromatographic peaks of the Dioscorea alata are attributed, and are respectively: peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is raffinose. According to the calculation results of the standard curve, the monosaccharide and oligosaccharide contents in the Dioscorea alata sample are calculated according to the dry product, and the results are shown in Table 6. Figure 3 )are attributed, and are respectively: peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is raffinose. According to the calculation results of the standard curve, the monosaccharide and oligosaccharide contents in the Dioscorea alata sample are calculated according to the dry product, and the results are shown in Table 6.

[0036] Table 6 Determination results of four sugar components in Dioscorea alata (n=3, %)

[0037] Experiment four The method of the present application is used for HPLC-CAD detection of cassava samples from different origins (a total of 6 batches, sample numbers Me1~Me6). 1 g of different cassava sample medicinal material powder (passed through a 4# sieve) is taken, placed in a 250 mL round-bottom flask, 100 mL of water is added, heated to reflux for 1.5 h, and filtered while hot. The residue is repeatedly extracted once according to the above method, and the two filtrates are combined. The filtrate is cooled, concentrated under reduced pressure to about 30 mL, then anhydrous ethanol is added to make the alcohol content of the solution reach 90%, shaken well, and placed at 4°C for 12 h. The supernatant is discarded, the precipitate is washed with anhydrous ethanol for 2 times and acetone for 1 time, and after each washing, it is centrifuged at 4000 r / min for 10 min and the supernatant is discarded. The precipitate is dried to no alcohol smell, and then vacuum freeze-dried to constant weight; 50 mg of freeze-dried powder is accurately added to 1 mL of distilled water, dissolved, and the cassava monosaccharide and oligosaccharide test solution is obtained.

[0038] The precise aspirate sample solution of the Dioscorea alata is injected into the HPLC-CAD chromatograph, and the absorbance of each sample is determined according to the method in item 2.3. The HPLC-CAD chromatographic peaks of the Dioscorea alata are attributed, and are respectively: peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is raffinose. According to the calculation results of the standard curve, the monosaccharide and oligosaccharide contents in the Dioscorea alata sample are calculated according to the dry product, and the results are shown in Table 6. Figure 3 )are attributed, and are respectively: peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is raffinose. According to the calculation results of the standard curve, the monosaccharide and oligosaccharide contents in the Dioscorea alata sample are calculated according to the dry product, and the results are shown in Table 6.

[0039] Table 7 Determination results of four sugar components in cassava (n=3, %)

[0040] Experiment five The method of the present application is used to detect different origin Dioscorea alata L. samples (5 batches in total, sample numbers Ib1~Ib5) by HPLC-CAD. 1 g of different Dioscorea alata L. sample medicinal material powder (passed through a No. 4 sieve) is placed in a 250 mL round bottom flask, 100 mL of water is added, heated reflux heating is performed for 1.5 h, and hot filtration is performed. The residue is repeatedly extracted once according to the above method, and the two filtrates are combined. The filtrate is cooled, concentrated under reduced pressure to about 30 mL, then anhydrous ethanol is added to make the alcohol content of the solution reach 90%, shaken, and placed at 4 DEG C for 12 h. The supernatant is discarded, the precipitate is washed with anhydrous ethanol twice and acetone once, each time after washing, centrifugation is performed at 4000 r / min for 10 min and the supernatant is discarded. The precipitate is dried until no alcohol smell is left, and then vacuum freeze-dried to a constant weight; 50 mg of the freeze-dried powder is precisely added to 1 mL of distilled water, dissolved, and then the Dioscorea alata L. monosaccharide and oligosaccharide test sample solution is obtained.

[0041] 10 μL of the Dioscorea alata L. test sample solution is precisely taken and injected into the HPLC-CAD chromatograph, the absorbance of each sample is determined according to the method in item 2.3, and the Dioscorea alata L. HPLC-CAD chromatographic peaks are attributed, which are as follows: peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is raffinose. Figure 3 According to the results of the standard curve, the monosaccharide and oligosaccharide contents in the Dioscorea alata L. samples are calculated, and the results are shown in Table 8.

[0042] Table 8: Determination results of four sugar components in Dioscorea alata L. (n=3, %)

[0043] Compared with the prior art, the present application has the following beneficial technical effects: (1) the present application develops a method for distinguishing Chinese medicinal material iron stick yam and its counterfeit based on HPLC-CAD, finds characteristic peaks that can distinguish iron stick yam and its counterfeit, and can quickly and effectively identify the two; (2) the present application adopts an empirical identification method different from the conventional method based on the appearance of iron stick yam and its counterfeit, avoids subjective tolerance, enhances the objectivity and specificity of the method, and compared with the reported molecular identification, the operation is simple and the time is short; the present method can effectively solve the problem that iron stick yam and its counterfeit are difficult to distinguish in actual work, provides technical support and basis for medicinal material market management, quality and efficacy of iron stick yam, and market management, and has practical application value.

Claims

1. A method for constructing characteristic chromatograms to distinguish between Dioscorea opposita and its adulterants based on HPLC-CAD, characterized in that, This is achieved through the following steps: S1. Preparation of the test solution of Dioscorea opposita: Weigh an appropriate amount of genuine Dioscorea opposita, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, and add water in a volume that is 100 times the weight of the medicinal material each time. The weight and volume refer to g for solids and mL for liquids. Heat and reflux to extract 1-2 times, each extraction time being 1-2 hours. Combine the extracts, cool to room temperature, concentrate under reduced pressure to 30% of the added water volume, then add anhydrous ethanol to make the alcohol content of the solution reach 85%-95% by volume. Shake well, let stand at 4℃ for 12 hours, and discard the supernatant. Wash the precipitate with anhydrous ethanol and acetone in sequence. After washing, centrifuge at 4000r for 10 minutes, discard the supernatant, obtain the precipitate, evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight, obtain powder, dissolve in distilled water to obtain the test solution of Dioscorea opposita. S2. Preparation of the adulterated medicinal material solution: Weigh an appropriate amount of adulterated medicinal material, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, each time adding water at a volume 100 times the weight of the medicinal material, heat and reflux to extract 1-2 times, each extraction time is 1-2 hours, combine the extracts, cool to room temperature, concentrate under reduced pressure to 30% of the added water volume, then add anhydrous ethanol to make the solution contain 85%~95% alcohol by volume, shake well, let stand at 4℃ for 12 hours, discard the supernatant; wash the precipitate with anhydrous ethanol and acetone in sequence, after washing, centrifuge at 4000r for 10min, discard the supernatant to obtain the precipitate, then evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight to obtain powder, add distilled water to dissolve, to obtain the adulterated medicinal material solution; The counterfeit medicinal materials are selected from Dioscorea opposita and Dioscorea glutinosa (both belonging to the Dioscoreaceae family), cassava (belonging to the Euphorbiaceae family), and Ipomoea spp. (belonging to the Convolvulaceae family). S3. Preparation of the reference solution: Weigh 8-22 mg of D-fructose, 13-20 mg of D-glucose, 20-20 mg of sucrose, 8-19 mg of fructosyl sucrose, and 5-18 mg of raffinose, dissolve in distilled water to prepare a solution with a mass concentration of 1-10 mg / mL. -1 The solution is then used to obtain a mixed reference solution; S4. The test solution, adulterant solution, and reference solution were detected using a high-performance liquid chromatography-electrospray ionization (HPLC-ESI) detector. The chromatographic conditions were as follows: an Asahipak NH2P-504E column (4.6 mm × 250 mm, 3.5 μm) was used at a column temperature of 40 ℃, with gradient elution at a flow rate of 1 mL / min. Acetonitrile A and water B were used as the mobile phase. The gradient elution program was as follows: 0–8 min, 75% A; 8–10 min, 75% A–71% A; 10–15 min, 71% A; 15–20 min, 71–69% A; 20–25 min, 69% A. A Cornoa VEO SD ESI detector was used for detection, with the following parameters: acquisition frequency 50 Hz, filter constant 2.0, and gas pressure 55.0 Pa. Based on the detection results, standard characteristic chromatograms of the Chinese yam, its adulterant, and the reference solution were plotted. The standard characteristic spectrum of the iron yam includes 5 characteristic peaks: peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, peak 4 is sucrose trisaccharide, and peak 5 is raffinose. The standard characteristic spectrum of the counterfeit product includes four characteristic peaks: peak 1 is D-fructose, peak 2 is D-glucose, peak 3 is sucrose, and peak 4 is sucrose trisaccharide.

2. The method for constructing characteristic chromatograms to distinguish between Dioscorea opposita and its adulterants based on HPLC-CAD according to claim 1, characterized in that, This is achieved through the following steps: S1. Preparation of the test solution of Dioscorea opposita: Weigh 1g of genuine Dioscorea opposita, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add 100ml of pure water, heat and reflux to extract twice, each extraction time is 2h, combine the extracts, cool to room temperature, concentrate under reduced pressure to 30ml, then add anhydrous ethanol to make the alcohol volume of the solution reach 85%, shake well, let stand at 4℃ for 12h, discard the supernatant; wash the precipitate with anhydrous ethanol and acetone in turn, after washing, centrifuge at 4000r for 10min, discard the supernatant, obtain the precipitate, evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight, obtain powder, add distilled water to dissolve, and obtain the test solution of Dioscorea opposita. S2. Preparation of the adulterated medicinal material solution: Weigh 1g of adulterated medicinal material, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, 100ml each time, heat and reflux to extract twice, each extraction time is 2h, combine the extracts, cool to room temperature, concentrate under reduced pressure to 30ml, then add anhydrous ethanol to make the alcohol volume of the solution reach 85%, shake well, let stand at 4℃ for 12h, discard the supernatant; wash the precipitate with anhydrous ethanol and acetone in sequence, after washing, centrifuge at 4000r for 10min, discard the supernatant to obtain the precipitate, evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight to obtain powder, add distilled water to dissolve, and obtain the adulterated medicinal material solution; S3. Preparation of reference solutions: Weigh 21.40 mg D-fructose, 19.45 mg D-glucose, 19.55 mg sucrose, 15.90 mg fructotriose, and 17.55 mg raffinose, dissolve in distilled water to prepare solutions with mass concentrations of 4.28, 3.89, 3.91, 3.18, and 3.51 mg / mL. -1 The solution was used to obtain a mixed reference solution; S4. Detect the test solution, adulterant medicinal material solution, and reference solution using a high-performance liquid chromatography-electrospray detector. Based on the detection results, draw standard characteristic chromatograms of the iron yam reference medicinal material and its adulterant reference medicinal material.

3. The method for constructing characteristic chromatograms to distinguish between Dioscorea opposita and its adulterants based on HPLC-CAD according to claim 1, characterized in that, This is achieved through the following steps: S1. Preparation of the test solution of Dioscorea opposita: Weigh 2g of genuine Dioscorea opposita, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, 200ml each time, heat and reflux for 2h, extract twice, combine the filtrates, cool the extract to room temperature, concentrate it under reduced pressure to 60ml, then add anhydrous ethanol to make the alcohol content of the solution reach 95% by volume, shake well, let it stand at 4℃ for 12h, and discard the supernatant; wash the precipitate with anhydrous ethanol and acetone in sequence, after washing, centrifuge at 4000r for 10min, discard the supernatant, obtain the precipitate, evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight, obtain powder, add distilled water to dissolve, and obtain the test solution of Dioscorea opposita. S2. Preparation of the adulterated medicinal material solution: Weigh 2g of adulterated medicinal material, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, 200ml each time, heat and reflux for 2h, extract twice, combine the filtrates, cool the extract to room temperature, combine the extracts, cool to room temperature, concentrate under reduced pressure to 60ml, then add anhydrous ethanol to make the alcohol content of the solution reach 95% by volume, shake well, let stand at 4℃ for 12h, discard the supernatant; wash the precipitate with anhydrous ethanol and acetone in sequence, after washing, centrifuge at 4000r for 10min, discard the supernatant, obtain the precipitate, evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight, obtain powder, add distilled water to dissolve, and obtain the adulterated medicinal material solution; S3. Preparation of reference solutions: Weigh 12.56 mg D-fructose, 15.78 mg D-glucose, 15.82 mg sucrose, 18.36 mg fructosyl sucrose, and 17.02 mg raffinose, dissolve in distilled water to prepare solutions with mass concentrations of 6.28, 7.89, 7.91, 9.18, and 8.51 mg / mL. -1 The solution was used to obtain a mixed reference solution; S4. Detect the test solution, adulterant medicinal material solution, and reference solution using a high-performance liquid chromatography-electrospray detector. Based on the detection results, draw standard characteristic chromatograms of the iron yam reference medicinal material and its adulterant reference medicinal material.

4. The method for constructing characteristic chromatograms to distinguish between Dioscorea opposita and its adulterants based on HPLC-CAD according to claim 1, characterized in that, This is achieved through the following steps: S1. Preparation of the test solution of Dioscorea opposita: Weigh 5g of genuine Dioscorea opposita, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, 500mL each time, heat and reflux for 2h, extract twice, combine the filtrates, cool the extract to room temperature, concentrate it under reduced pressure to 150mL, then add anhydrous ethanol to make the alcohol content of the solution reach 90% by volume, shake well, let it stand at 4℃ for 12h, and discard the supernatant; wash the precipitate with anhydrous ethanol and acetone in sequence, after washing, centrifuge at 4000r for 10min, discard the supernatant, obtain the precipitate, evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight, obtain powder, add distilled water to dissolve, and obtain the test solution of Dioscorea opposita. S2. Preparation of the adulterated medicinal material solution: Weigh 5g of adulterated medicinal material, dry and pulverize it, pass it through a 60-mesh sieve, place it in a round-bottom flask, add pure water, 500ml each time, heat and reflux for 2h, extract twice, combine the filtrates, cool the extract to room temperature, combine the extracts, cool to room temperature, concentrate under reduced pressure to 150ml, then add anhydrous ethanol to make the alcohol content of the solution reach 90% by volume, shake well, let stand at 4℃ for 12h, discard the supernatant; wash the precipitate with anhydrous ethanol and acetone in sequence, after washing, centrifuge at 4000r for 10min, discard the supernatant, obtain the precipitate, evaporate to dryness until there is no alcohol odor, freeze dry under vacuum to constant weight, obtain powder, dissolve in distilled water to obtain the adulterated medicinal material solution; S3. Preparation of reference solutions: Weigh 8.56 mg D-fructose, 13.78 mg D-glucose, 15.82 mg sucrose, 8.36 mg fructotriose, and 5.02 mg raffinose, dissolve in distilled water to prepare solutions with mass concentrations of 4.28, 6.89, 7.91, 4.18, and 2.51 mg / mL. -1 The solution was used to obtain a mixed reference solution; S4. Detect the test solution, adulterant medicinal material solution, and reference solution using a high-performance liquid chromatography-electrospray detector. Based on the detection results, draw standard characteristic chromatograms of the iron yam reference medicinal material and its adulterant reference medicinal material.

5. The application of the characteristic spectrum constructed by any one of claims 1-4 in the quality detection, evaluation and control of Dioscorea opposita, or in distinguishing Dioscorea opposita from its adulterants.

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