Peucedanum praeruptorum dunn and counterfeit product fingerprint spectrum identification method and application thereof

Abstract

The invention relates to the technical field of analysis and detection, and particularly discloses an identification method and application of fingerprints of radix peucedani and counterfeit products thereof. The method comprises the following steps: preparing a reference solution and a test solution, and carrying out ultra-high performance liquid chromatography under the chromatographic conditions that a mobile phase A is 60% methanol and 40% tetrahydrofuran, a mobile phase B is ultrapure water, gradient elution is carried out for 0-25 minutes, 40%-65% of the mobile phase A and 60%-35% of the mobile phase B to establish the fingerprint spectrum. The method can effectively distinguish peucedanum praeruptorum (hard peucedanum praeruptorum, pilose peucedanum praeruptorum and cloud peucedanum praeruptorum), can judge peucedanum praeruptorum adulterated peucedanum praeruptorum adulterated peucedanum praeruptorum with the adulteration rate of 30% or above, and can effectively distinguish genuine peucedanum praeruptorum from fake

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to an identification method and application of the fingerprint of Peucedanum praeruptorum Dunn and its counterfeits. Background Art

[0002] Peucedanum praeruptorum Dunn is the dried root of the plant Peucedanum praeruptorum Dunn of the Umbelliferae family, and has the effects of lowering qi and resolving phlegm, dispersing wind and clearing heat. The local Chinese medicinal material standards in many regions have respectively included plants of the genus Peucedanum or Ligusticum of the Umbelliferae family such as Peucedanum praeruptorum Dunn var. rigidum, Peucedanum praeruptorum Dunn var. villosum, and Peucedanum praeruptorum Dunn var. rubricaule as the common substitutes for Peucedanum praeruptorum Dunn. However, many merchants, intentionally or unintentionally, sell the common substitutes for Peucedanum praeruptorum Dunn as Peucedanum praeruptorum Dunn, or adulterate the common substitutes for Peucedanum praeruptorum Dunn in Peucedanum praeruptorum Dunn, resulting in the chaos of the Peucedanum praeruptorum Dunn varieties in the market and further exacerbating the problems of the safety of Peucedanum praeruptorum Dunn in medication and the quality of products. This makes the identification method of traditional Chinese medicine Peucedanum praeruptorum Dunn particularly important. However, since Peucedanum praeruptorum Dunn and its counterfeits are mostly plants of the same family and genus, the medicinal properties and microscopic characteristics are similar, and the Peucedanum praeruptorum Dunn - related medicinal materials in the market mostly circulate in the form of cut pieces, which further increases the difficulty of identifying Peucedanum praeruptorum Dunn.

[0003] The traditional identification methods of Peucedanum praeruptorum Dunn mainly rely on morphological identification, microscopic identification and physicochemical identification, and multiple methods need to be used in combination when necessary. Morphological identification mainly observes the whole medicinal materials and cut pieces of Peucedanum praeruptorum Dunn. Affected by the subjective initiative of the appraisers, the identification results are often interfered by human factors, and high requirements are imposed on the medicinal properties of the materials; in actual research, microscopic identification has a high cost, the instruments are expensive, and the appraisers need to have skills in plant anatomy, plant microscopic section preparation technology and proficient operation skills of relevant microscopic instruments; Peucedanum praeruptorum Dunn contains a variety of complex chemical components. Sometimes, using a few physicochemical characteristics as the basis for identification cannot achieve the identification effect, and physicochemical identification usually requires complex pretreatment of the medicinal materials, which is time - consuming and laborious. For example, Huang Donglan et al. (Rapid Identification of Peucedanum praeruptorum Dunn and Its Counterfeit Saposhnikovia divaricata (Turcz.) Schischk. by Infrared Second - Derivative Spectroscopy. Journal of Shaoguan University [J]. 2009, 12) compared the infrared spectra of Peucedanum praeruptorum Dunn and Saposhnikovia divaricata (Turcz.) Schischk. through one - dimensional infrared spectroscopy technology. Although the purpose of quickly identifying Peucedanum praeruptorum Dunn and Saposhnikovia divaricata (Turcz.) Schischk. can be achieved, it has high requirements for the operators, and the detection results are greatly limited by the environment and the subjective initiative of people. Therefore, the traditional identification methods are difficult to simply, quickly and accurately distinguish the counterfeits of Peucedanum praeruptorum Dunn effectively.

[0004] Hu Yijuan (Research on HPLC Fingerprint of Peucedanum praeruptorum Dunn [J]. China Journal of Traditional Chinese Medicine and Pharmacy. 2012, 19(5): 437-439) conducted a fingerprint study on Peucedanum praeruptorum Dunn. However, only 6 common peaks were obtained by its HPLC detection method, and a large number of peaks in the characteristic chromatogram were separated in a short time, resulting in an uneven baseline, inaccurate analysis of the active ingredients in Peucedanum praeruptorum Dunn, and affecting the quality determination. Chinese Patent Application CN110297045A discloses a method for detecting the characteristic chromatogram of Peucedanum praeruptorum Dunn formula granules, which can simultaneously detect praeruptorin A and praeruptorin B, can identify 8 characteristic peaks, and can be used for the quality detection of formula granules, reflecting the overall appearance of multiple components of formula granules. However, the number of characteristic peaks of this method is relatively small, the resolution and symmetry of individual characteristic peaks (such as praeruptorin B) are relatively poor, and the analysis time is relatively long, so it cannot be applied to the rapid identification of Peucedanum praeruptorum Dunn and its counterfeits.

[0005] Therefore, there is an urgent need for an identification method that can quickly, accurately and simply distinguish Peucedanum praeruptorum Dunn and its counterfeits. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides an identification method and application of the fingerprint of Peucedanum praeruptorum Dunn and its counterfeits. This method can effectively distinguish genuine Peucedanum praeruptorum Dunn from its counterfeits, can judge the adulterated Peucedanum praeruptorum Dunn with an adulteration rate of more than 30%, and can more quickly, accurately and comprehensively distinguish the counterfeits of Peucedanum praeruptorum Dunn compared with traditional identification methods.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] On the one hand, the present invention provides an identification method of the fingerprint of Peucedanum praeruptorum Dunn and its counterfeits, including the following steps:

[0009] S1. Preparation of the reference substance solution: Take praeruptorin II, praeruptorin A, praeruptorin B and praeruptin E of Peucedanum praeruptorum Dunn respectively to prepare a mixed reference substance solution;

[0010] S2. Preparation of the test solution: Take the test sample, extract it with methanol and filter to obtain the test solution;

[0011] S3. Ultra-high performance liquid chromatography determination: Inject the mixed reference substance solution and the test solution into the liquid chromatograph respectively. The chromatographic conditions include: mobile phase A is 60% methanol - 40% tetrahydrofuran, mobile phase B is ultrapure water, and gradient elution is 0-25 min, 40% → 65% mobile phase A, 60% → 35% mobile phase B;

[0012] S4. Establishment of the fingerprint: Record the results of the ultra-high performance liquid chromatography determination and establish the fingerprint.

[0013] Preferably, the chromatographic conditions in step S3 further include: the chromatographic column is ACQUITY BEH C18 column, ACQUITY any one of HSS T3 column and Phenomenex SuperLu C18-AQ column.

[0014] More preferably, the chromatographic column is ACQUITY BEH C18 column.

[0015] Preferably, the chromatographic conditions in step S3 further include: the column temperature is 25 - 35 °C; the flow rate is 0.2 mL / min - 0.3 mL / min; the wavelength is 310 nm - 322 nm.

[0016] More preferably, the column temperature is 30 °C; the flow rate is 0.25 mL / min; the wavelength is 321 nm.

[0017] Preferably, the test sample in step S2 includes at least one of Peucedani Radix, Peucedani rigidum, Peucedani pubescens, and Peucedani decursivum.

[0018] More preferably, the Peucedani Radix includes any one of Peucedani Radix medicinal materials and Peucedani Radix decoctions; the Peucedani rigidum includes any one of Peucedani rigidum medicinal materials and Peucedani rigidum decoctions; the Peucedani pubescens includes any one of Peucedani pubescens medicinal materials and Peucedani pubescens decoctions; the Peucedani decursivum includes any one of Peucedani decursivum medicinal materials and Peucedani decursivum decoctions.

[0019] Specifically, the mixed reference substance solution in step S1 needs to be diluted with methanol.

[0020] Preferably, the extraction in step S2 is ultrasonic extraction.

[0021] Preferably, the conditions of the ultrasonic extraction include: power 200 - 300 W, frequency 40 - 60 kHz, time 5 - 15 minutes.

[0022] More preferably, the conditions of the ultrasonic extraction include: power 250 W, frequency 50 kHz, time 10 minutes.

[0023] Preferably, the filtration in step S2 is through a 0.22 μm microporous membrane filter.

[0024] Preferably, the mass - volume ratio of the test sample to methanol in step S2 is 0.1 - 0.15 g:10 mL.

[0025] Preferably, the mass - volume ratio of the test sample to methanol is 0.1 g:10 mL.

[0026] Preferably, the volume of the mixed reference substance solution in step S3 is 1 - 3 μl, and the volume of the test sample solution is 1 - 3 μl.

[0027] More preferably, the volume of the mixed reference substance solution is 2 μl, and the volume of the test sample solution is 2 μl.

[0028] On the other hand, the present invention also provides an application of the identification method of the fingerprint of Peucedanum praeruptorum Dunn and its counterfeits in distinguishing Peucedanum praeruptorum Dunn from its counterfeits.

[0029] Preferably, the counterfeits of Peucedanum praeruptorum Dunn include mixed counterfeits and adulterated products of Peucedanum praeruptorum Dunn.

[0030] Specifically, the mixed counterfeits of Peucedanum praeruptorum Dunn include at least one of pure Peucedanum harry-smithii Fedde, pure Peucedanum pubescens (Bl.) Benth., and pure Peucedanum rubricaule Shan & Sheh; the adulterated product of Peucedanum praeruptorum Dunn is the pure Peucedanum harry-smithii Fedde, pure Peucedanum pubescens (Bl.) Benth., or pure Peucedanum rubricaule Shan & Sheh added to the genuine Peucedanum praeruptorum Dunn, and the added mass is greater than or equal to 30% and less than 100% of the mass of the genuine Peucedanum praeruptorum Dunn.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention constructs a reference fingerprint of genuine Peucedanum praeruptorum Dunn by Ultra Performance Liquid Chromatography (UPLC), which can effectively distinguish the counterfeits of Peucedanum praeruptorum Dunn (Peucedanum harry-smithii Fedde, Peucedanum pubescens (Bl.) Benth., and Peucedanum rubricaule Shan & Sheh); from the similarity results of the fingerprints of adulterated products with different proportions and the reference fingerprint of Peucedanum praeruptorum Dunn, it can be seen that the identification method of the present invention can judge the adulterated products of Peucedanum praeruptorum Dunn with an adulteration rate of more than 30%; and the identification method of the present invention has better durability, and the obtained fingerprint is more delicate. Compared with traditional identification methods, it can more quickly, accurately and comprehensively achieve the effective distinction between Peucedanum praeruptorum Dunn and its counterfeits. Description of the Drawings

[0033] Figure 1 It is the UPLC diagram of the mixed reference substance in Example 1, where peak 3 is praeruptorin II, peak 5 is praeruptorin A, peak 12 is praeruptorin B, and peak 14 is praeruptin E;

[0034] Figure 2 It is the UPLC diagram of the test sample of Peucedanum praeruptorum Dunn (Q1) in Example 1, where peak 3 is praeruptorin II, peak 5 is praeruptorin A, peak 12 is praeruptorin B, and peak 14 is praeruptin E;

[0035] Figure 3 It is the superposition fingerprint and the standard fingerprint of Peucedanum praeruptorum Dunn in Example 1;

[0036] Figure 4It is the overlapping fingerprint spectrum of Peucedanum praeruptorum Dunn and its standard fingerprint spectrum in Example 1;

[0037] Figure 5 It is the overlapping fingerprint spectrum of Peucedanum pubescens Maxim. and its standard fingerprint spectrum in Example 1;

[0038] Figure 6 It is the overlapping fingerprint spectrum of Peucedanum rubricaule Shan et Sheh and its standard fingerprint spectrum in Example 1;

[0039] Figure 7 It is the graph showing the influence of the adulteration rate on the similarity of the fingerprint spectrum of Peucedanum praeruptorum Dunn in Example 1;

[0040] Figure 8 It is the graph showing the investigation results of different brands of chromatographic columns in Example 1;

[0041] Figure 9 It is the graph showing the investigation results of the column temperature in Example 1;

[0042] Figure 10 It is the graph showing the investigation results of the flow rate in Example 1;

[0043] Figure 11 It is the UPLC diagram of the test sample of Peucedanum praeruptorum Dunn (Q1) and the mixed reference substance in Comparative Example 1, where peak 1 is praeruptorin II, peak 2 is praeruptorin A, peak 3 is praeruptorin B, and peak 4 is praeruptin E;

[0044] Figure 12 It is the UPLC diagram of the test sample of Peucedanum praeruptorum Dunn (Q1) and the mixed reference substance in Comparative Example 2, where peak 1 is praeruptorin II, peak 2 is praeruptorin A, peak 3 is praeruptorin B, and peak 4 is praeruptin E. Specific embodiments

[0045] The present invention will be described below through specific examples to make the technical solutions of the present invention easier to understand and master. However, the present invention is not limited thereto. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0046] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein. Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" as used herein include both singular and plural referents. Numerical ranges expressed by endpoints include all values and fractions within the corresponding ranges, as well as the expressed endpoints.

[0047] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. For the experimental methods described in the following embodiments, unless otherwise specified, they are all conventional methods; for the reagents and materials, unless otherwise specified, they can all be obtained from commercial channels.

[0048] Experimental Instruments and Drugs

[0049] Instruments: Waters Acquity H-Class ultra-high performance liquid chromatograph (Waters Corporation, USA); MS105DU electronic analytical balance (Mettler-Toledo Technology Co., Ltd.); KQ-300DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); electrothermal blast drying oven; ACQUITY BEH C18 chromatographic column (1.7μm, 2.1mm×100mm); ACQUITY HSS T3 (1.8um, 2.1mm×100mm); Phenomenex SuperLu C18-AQ (1.8um, 2.1mm×100mm).

[0050] Drugs: Praeruptorin A (content: 99.4%, batch number: 111711-201904), Praeruptorin B (content: 99.7%, batch number: 111904-202105) were purchased from the National Institutes for Food and Drug Control; Praeruptorin II (content: 98.0%, batch number: PS220407-03), Praeruptorin E (content: 98.0%, batch number: PS001175) were purchased from Chengdu Pusi Biotechnology Co., Ltd. Methanol and tetrahydrofuran are of chromatographic grade, water is ultrapure water, and other reagents are all of analytical grade.

[0051] Experimental Materials

[0052] Details of the samples used in the experiment are shown in Table 1, and the collection time was from January 2020 to October 2023. And they were identified by Researcher Yujinbao of Jiangxi Academy of Traditional Chinese Medicine. Q1-Q32 are Peucedanum praeruptorum Dunn, Y1-Y12 are Peucedanum harry-smithii Fedde var. subglabrum Shan et Sheh, M1-M9 are Ligusticum brachylobum Franch., and H1-H15 are Peucedanum rubricaule Shan et Sheh.

[0053] Table 1 Sample Information Table

[0054]

[0055]

[0056]

[0057] Example 1

[0058] 1. Chromatographic conditions: ACQUITY BEH C18 chromatographic column (1.7 μm, 2.1 mm × 100 mm); mobile phase A is methanol - tetrahydrofuran (60:40), mobile phase B is ultrapure water, gradient elution (0 - 25 min, 40% → 65% mobile phase A, 60% → 35% mobile phase B); detection wavelength is 321 nm, column temperature is 30 °C, flow rate is 0.25 mL / min, injection volume is 2 μL.

[0059] 2. Preparation of solutions:

[0060] (1) Preparation of mixed reference substance solution

[0061] Precisely weigh 9.68 mg of praeruptorin II, 11.60 mg of praeruptorin A, 11.34 mg of praeruptorin B, and 10.38 mg of praeruptorin E respectively, place them in 4 25 - mL volumetric flasks, dilute to the mark with methanol, and mix well to obtain the stock solution of mixed reference substances. Precisely measure 1 mL of the stock solutions of the 4 reference substances into the same 10 - mL volumetric flask to obtain a mixed reference substance solution with concentrations of 37.9, 46.1, 45.2, and 40.7 μg / mL respectively.

[0062] (2) Preparation of test solution

[0063] Take about 0.1 g of the powder of this product (passed through No. 3 sieve), precisely weigh it, place it in a stoppered conical flask, precisely add 10 mL of methanol, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 50 kHz) for 10 minutes, let it cool and then weigh it again, make up the lost weight with methanol, shake well, filter through a 0.22 - μm microporous filter membrane, and take the subsequent filtrate, that is, obtain the test solution.

[0064] 3. Establishment of fingerprint

[0065] Take Peucedanum praeruptorum Dunn (Q1 - Q32), Peucedanum harry-smithii Fedde (Y1 - Y12), Peucedanum pubescens (M1 - MQ09), and Peucedanum rubricaule Shan et Sheh (H1 - H15) respectively, prepare the test solutions according to the above "preparation method of test solution", inject samples for determination under the above "chromatographic conditions", and record the chromatograms (as shown in Figure 1 and Figure 2 ). Import the chromatographic data of Peucedanum praeruptorum Dunn (Q1 - Q20), Peucedanum harry-smithii Fedde (Y1 - Y12), Peucedanum pubescens (M1 - MQ09), and Peucedanum rubricaule Shan et Sheh (H1 - H15) into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" respectively, set Q1, Y1, M1, and H1 as the reference chromatograms respectively, select the median method for the standard chromatogram generation method, set the time window width to 0.1 min, perform full-spectrum peak matching after multi-point calibration, and obtain the standard chromatograms R and the superposed chromatograms of Peucedanum praeruptorum Dunn, Peucedanum harry-smithii Fedde, Peucedanum pubescens, and Peucedanum rubricaule Shan et Sheh respectively (see Figures 3 - 6), and save the standard spectrum R of Peucedani Radix.

[0066] Results:

[0067] From the peak matching results, it can be seen that Peucedani Radix, Peucedanum rigidum, Peucedanum pubescens, and Peucedanum rubricaule respectively determined 16, 13, 17, and 12 common peaks. Among them, the peak areas of peak 12 (praeruptorin B) and peak 31 (unknown peak) were moderate and the resolution was good. Therefore, peak 12 was selected as the reference peak for Peucedani Radix, Peucedanum rigidum, and Peucedanum pubescens, and peak 31 was selected as the reference peak for Peucedanum rubricaule. The similarities of 20 batches of Peucedani Radix with its standard spectrum were: 0.998, 0.993, 0.974, 0.991, 0.997, 0.999, 0.995, 0.984, 0.992, 0.997, 0.995, 1.000, 0.995, 0.999, 1.000, 0.919, 0.984, 0.997, 0.987, 0.992, all ≥ 0.919; the similarities of 12 batches of Peucedanum rigidum with its standard spectrum were: 0.980, 0.989, 0.953, 0.983, 0.976, 0.988, 0.988, 0.993, 0.993, 0.979, 0.993, 0.993, all ≥ 0.953; the similarities of 9 batches of Peucedanum pubescens with its standard spectrum were: 0.989, 0.917, 0.982, 0.974, 0.987, 0.986, 0.982, 0.974, 0.920, all ≥ 0.917; the similarities of 15 batches of Peucedanum rubricaule with its standard spectrum were: 0.999, 0.995, 0.991, 1.000, 1.000, 0.996, 0.992, 0.999, 0.995, 0.986, 0.993, 0.999, 0.990, 0.994, 0.991, all ≥ 0.986, indicating that the differences between batches of samples were small.

[0068] By comparing with reference substances, 4 components were identified, namely peucedanocoumarin II (peak 3), praeruptorin A (peak 5), praeruptorin B (peak 12), and praeruptorin E (peak 14). There were 9 common peaks between Peucedanum rigidum and Peucedani Radix, namely peaks 1, 2, 3, 8, 10, 11, 12, 14, and 16; there were 10 common peaks between Peucedanum pubescens and Peucedani Radix, namely peaks 1, 3, 4, 5, 8, 9, 10, 11, 12, and 14; there were no common peaks between Peucedanum rubricaule and Peucedani Radix. It can be seen from this that the chemical compositions of Peucedanum rigidum, Peucedanum pubescens, and Peucedani Radix are similar, while the chemical composition of Peucedanum rubricaule is quite different from that of Peucedani Radix.

[0069] 4. Methodology investigation:

[0070] Take Peucedani Radix (Q1), prepare the test solution according to the "Preparation Method of Test Solution" in Example 1 above, inject and determine under the "Chromatographic Conditions" in Example 1 above, inject continuously for 6 times, and record the chromatogram. Among them, taking the 12th peak (praeruptorin B) of Peucedani Radix as the reference peak, the RSD of the relative retention time of the common peaks is less than 0.62%, and the RSD of the relative peak area is less than 1.83%, indicating good instrument precision.

[0071] Take Peucedani Radix (Q1) of the same batch, prepare the test solution according to the "Preparation Method of Test Solution" in Example 1 above, inject and determine under the "Chromatographic Conditions" in Example 1 above, and the calculated RSD of the relative retention time of the common peaks is less than 0.56%, and the RSD of the relative peak area is less than 1.24%, indicating good repeatability of this method.

[0072] The Peucedani Radix test solution prepared according to the "Preparation Method of Test Solution" in Example 1 above was placed at room temperature for 0, 4, 8, 12, 18, and 24 h respectively, injected and determined under the "Chromatographic Conditions" in Example 1 above, and the calculated RSD of the relative retention time of the common peaks is less than 0.32%, and the RSD of the relative peak area is less than 1.66%, indicating good stability of the test solution prepared by this method.

[0073] 5. Similarity comparison of different Peucedani Radix medicinal materials

[0074] According to the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition), select "Analysis and Inspection" in the working state, import the chromatographic data of the Peucedani Radix standard map R and Peucedani Radix (Q21-Q32), Peucedani praeruptorum Dunn (Y01-Y12), Peucedani pubescens (M01-MQ09), and Peucedani decursivum (Miq.) Maxim. (H01-H15) respectively. Taking the Peucedani Radix standard map as the reference chromatogram, using the median method, the time window is selected as 0.1 min, and after multi-point correction and automatic full-spectrum peak matching, calculate the similarity, and the results are shown in Table 2.

[0075] Table 2 Similarity of different Peucedani Radix medicinal materials and the Peucedani Radix standard map

[0076]

[0077] As can be seen from Table 2, the similarity of 12 batches of Peucedani Radix samples and the Peucedani Radix standard map is relatively high, ranging from 0.949 to 0.997, indicating that the fingerprint differences of Peucedani Radix from different origins and cultivation methods are not significant; the similarity of Peucedani praeruptorum Dunn, Peucedani pubescens, and Peucedani decursivum (Miq.) Maxim. to the Peucedani Radix standard map is between 0.070 and 0.240, indicating that there are significant differences in the fingerprint similarity between the adulterants and genuine products of Peucedani Radix, and the similarity can effectively distinguish Peucedani Radix and its adulterants.

[0078] 6. Influence of adulteration rate on the similarity of Peucedani Radix fingerprint

[0079] In order to investigate the effect of adulteration of Peucedanum hupensis, Peucedanum pubescens and Peucedanum cloud on the similarity of fingerprint spectra, Peucedanum hupensis (Q1) was adulterated with Peucedanum hupensis (Y1), Peucedanum pubescens (M1) and Peucedanum cloud (H1) at mass percentages of 0%, 10%, 20%, 30%, 40%, 50%, 70%, 90% and 100%, respectively. The similarity between different adulterated Peucedanum samples and Peucedanum standard spectra was calculated. The results are shown in Table 1. Figure 7 The results show that with the increase of adulteration rate, the similarity of samples gradually decreases. When 30% of hard peucedanum, hairy peucedanum and cloud peucedanum are added to peucedanum, the similarity decreases to 0.886, 0.871 and 0.857 respectively. It is preliminarily believed that the similarity of fingerprint spectra can be used to judge the adulterated peucedanum with an adulteration rate of more than 30%.

[0080] In summary, the results of fingerprint similarity determination show that when the sample similarity is less than 0.3, it indicates that the sample is a Peucedanum counterfeit; when 0.3 < similarity < 0.9, it indicates that the sample adulteration rate is ≥ 30%. That is, the constructed Peucedanum UPLC fingerprint can be used to identify common Peucedanum counterfeits and determine Peucedanum adulterants with an adulteration rate of more than 30%.

[0081] 7. Durability inspection

[0082] (1) Chromatographic column inspection

[0083] Peucedanum peucedanum (Q1) was used as the product, and the chromatographic column ACQUITY BEHC18 column (1.7 μm, 2.1 mm × 100 mm), ACQUITY HSS T3 (1.8um, 2.1×100mm) and Pheromon SuperLu C18-AQ (1.8um, 2.1×100mm) were investigated. The results are shown in Figure 8 , Table 3 and Table 4.

[0084] Table 3 Column durability investigation - relative retention time ratio

[0085]

[0086]

[0087] Table 4 Column durability investigation - relative peak area ratio

[0088]

[0089] The results showed that the peaks of the common peaks were relatively symmetrical and had good separation when the three chromatographic columns were used to detect the samples. The RSD of the relative retention time of the common peaks was between 0.26% and 6.07%, and the RSD of the relative peak area of ​​the common peaks was between 0.66% and 8.75%.

[0090] (2) Column temperature investigation

[0091] Take Peucedanum praeruptorum Dunn (Q1) as this product, and according to the method of Example 1 above, investigate the column temperatures of 25 °C, 30 °C, and 35 °C respectively. The results are shown in Figure 9 , Table 5 and Table 6.

[0092] Table 5 Column temperature investigation — Relative retention time ratio

[0093]

[0094]

[0095] Table 6 Column temperature investigation — Relative peak area ratio

[0096]

[0097] The results show that when the column temperature is between 25 - 35 °C, the peak shapes in the chromatogram are relatively symmetrical, the resolution is good, and the RSD of the relative retention time of the common peaks is less than 3%, indicating good method durability.

[0098] (3) Flow rate investigation

[0099] Take Peucedanum praeruptorum Dunn (Q1) as this product, and according to the method of Example 1 above, investigate the flow rates of 0.2 mL / min, 0.25 mL / min, and 0.3 mL / min respectively. The results are shown in Figure 10 , Table 7 and Table 8.

[0100] Table 7 Flow rate investigation — Relative retention time ratio

[0101]

[0102] Table 8 Flow rate investigation — Relative peak area ratio

[0103]

[0104] The results show that when the flow rates are 0.2 mL / min, 0.25 mL / min, and 0.3 mL / min respectively, the RSD of the relative retention time of each common peak is 0.37 - 7.31%. When the flow rate is 0.25 mL / min, the peak shape in the chromatogram is good and the resolution is moderate.

[0105] Comparative Example 1

[0106] Compared with Example 1, only the chromatographic conditions are different:

[0107] Chromatographic conditions: Agilent ZORBAX SB C18 (5μm, 4.6mm×250mm); mobile phase A is methanol, mobile phase B is ultrapure water, gradient elution (0 - 20 min, 15% → 45% A; 20 - 65 min, 45% → 95% A; 65 - 70 min, 95% A); detection wavelength is 321 nm, column temperature is 30 °C, flow rate is 1.0 mL / min, injection volume is 2 μL.

[0108] The results are as Figure 11 shown. This method has relatively few characteristic peaks, and the resolution and symmetry of individual characteristic peaks (peucedanin) are poor, and the analysis time is relatively long (70 min), so it cannot be applied to the rapid identification of Peucedani Radix and its counterfeits.

[0109] Comparative Example 2

[0110] Compared with Example 1, only the chromatographic conditions are different:

[0111] Chromatographic conditions: ACQUITY BEH C18 chromatographic column (1.7μm, 2.1mm×100mm); mobile phase A is methanol, mobile phase B is ultrapure water, gradient elution (0 - 30 min, 45% → 80% mobile phase A); detection wavelength is 321 nm, column temperature is 30 °C, flow rate is 0.25 mL / min, injection volume is 2 μL.

[0112] The results are as Figure 12 shown. This method has poor durability and cannot effectively separate the index components, praeruptorin A and praeruptorin E; and the identified characteristic peaks are few, so it cannot accurately and effectively identify genuine Peucedani Radix and its counterfeits.

[0113] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for identifying fingerprints of Peucedanum peucedanum and its counterfeits, characterized in that: The following steps are involved: S1. Preparation of reference solution: respectively taking peucedanum coumarin II, peucedanum acetylcholine A, peucedanum acetylcholine B and peucedanum acetylcholine E to prepare a mixed reference solution; S2. Preparation of test solution: extract the test sample with methanol and filter to obtain the test solution; S3. Ultra-high performance liquid chromatography determination: The mixed reference solution and the test solution were respectively injected into the liquid chromatograph. The chromatographic conditions included: mobile phase A was 60% methanol-40% tetrahydrofuran, mobile phase B was ultrapure water, and the gradient elution was 0-25 min, 40%→65% mobile phase A, 60%→35% mobile phase B; S4. Establishment of fingerprint: record the results of ultra-high performance liquid chromatography and establish the fingerprint.

2. The identification method according to claim 1, characterized in that: The chromatographic conditions described in step S3 also include: the chromatographic column is ACQUITY BEH C18 Column, ACQUITY Any of HSS T3 column and Pheromones SuperLuC18-AQ column.

3. The identification method according to claim 2, characterized in that: The chromatographic column is ACQUITY BEHC18 column.

4. The identification method according to claim 1, characterized in that: The chromatographic conditions described in step S3 also include: column temperature of 25-35° C.; flow rate of 0.2 mL / min-0.3 mL / min; wavelength of 310 nm-322 nm.

5. The identification method according to claim 4, characterized in that: The column temperature was 30° C., the flow rate was 0.25 mL / min, and the wavelength was 321 nm.

6. The identification method according to claim 1, characterized in that: The test sample described in step S2 includes at least one of the Chinese medicine Peucedanum chinense, Chinese medicine Peucedanum sclerosus, Chinese medicine Peucedanum pubescens and Chinese medicine Peucedanum yunnanensis.

7. The identification method according to claim 6, characterized in that: The Chinese medicine of Peucedanum chinense includes any one of Peucedanum chinense medicinal materials and Peucedanum chinense decoction pieces; the Chinese medicine of Peucedanum sibiricum includes any one of Peucedanum sibiricum medicinal materials and Peucedanum sibiricum decoction pieces; the Chinese medicine of Peucedanum pubescentum includes any one of Peucedanum pubescentum medicinal materials and Peucedanum pubescentum decoction pieces; the Chinese medicine of Peucedanum yunnanensis includes any one of Peucedanum yunnanensis medicinal materials and Peucedanum yunnanensis decoction pieces.

8. The identification method according to claim 1, characterized in that: The extraction described in step S2 is ultrasonic extraction.

9. The identification method according to claim 8, characterized in that: The conditions for the ultrasonic extraction include: power 200-300W, frequency 40-60kHz, and time 5-15 minutes.

10. The identification method according to claim 9, characterized in that: The conditions of the ultrasonic extraction include: power 250W, frequency 50kHz, and time 10 minutes.

11. The identification method according to claim 1, characterized in that: The mass volume ratio of the test sample to methanol in step S2 is 0.1-0.15 g:10 mL.

12. The identification method according to claim 11, characterized in that: The mass volume ratio of the test sample to methanol is 0.1 g:10 mL.

13. The identification method according to claim 1, characterized in that: The volume of the mixed reference solution in step S3 is 1-3 μl, and the volume of the test solution is 1-3 μl.

14. The identification method according to claim 13, characterized in that: The volume of the mixed reference solution is 2 μl, and the volume of the test solution is 2 μl.

15. Use of the identification method according to any one of claims 1 to 14 in distinguishing Peucedanum peucedanum from its counterfeit products.

16. The use according to claim 15, characterized in that: The peucedanum counterfeit products include peucedanum mixed counterfeit products and peucedanum adulterated products.

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