A method for identifying the fingerprint spectrum of peucedanum and its counterfeit and application

Abstract

The present application relates to the field of analytical detection technology, and specifically discloses a method for identifying the fingerprint spectrum of radix peucedani and its counterfeit products and application. The present application can effectively distinguish the counterfeit products (hard radix peucedani, hairy radix peucedani and radix peucedani yun) of radix peucedani, can determine the adulteration rate of the adulterated radix peucedani of more than 30%, and can more quickly, accurately and comprehensively realize the effective distinction between the genuine and counterfeit products of radix peucedani compared with the traditional identification methods, by preparing the reference solution and the sample solution, and by establishing the fingerprint spectrum through the chromatographic conditions of the ultra-high performance liquid chromatography, in which the mobile phase A is 60% methanol-40% tetrahydrofuran, the mobile phase B is ultrapure water, and the gradient elution is 40%→65% of the mobile phase A and 60%→35% of the mobile phase B within 0-25 min.

Description

Technical Field

[0001] This invention belongs to the field of analytical testing technology, specifically relating to a method and application for identifying fingerprint spectra of Peucedanum praeruptorum and its counterfeits. Background Technology

[0002] Peucedanum praeruptorum Dunn, a plant in the Apiaceae family, is the dried root of a plant with white flowers. It is used to lower qi, resolve phlegm, dispel wind, and clear heat. Many local standards for traditional Chinese medicine include various species of Peucedanum (such as hard-skinned Peucedanum, hairy Peucedanum, and cloud-skinned Peucedanum) from the Apiaceae family or the Ligusticum genus as traditional Peucedanum. However, many merchants intentionally or unintentionally sell traditional Peucedanum as white-flowered Peucedanum, or adulterate white-flowered Peucedanum with traditional Peucedanum, causing confusion in the market and exacerbating safety and quality issues related to its use. This makes the identification of Peucedanum particularly important. However, because Peucedanum and its adulterants are mostly from the same family and genus, their medicinal properties and microscopic characteristics are similar, and Peucedanum-like medicinal materials are mostly circulated in the form of processed slices, further increasing the difficulty of identification.

[0003] Traditional identification methods for Peucedanum praeruptorum mainly rely on morphological identification, microscopic identification, and physicochemical identification, sometimes requiring a combination of methods. Morphological identification primarily involves observing the whole medicinal material and its processed slices. However, the results are often influenced by human factors due to the subjective initiative of the identification worker, and it also requires high standards for the characteristics of the medicinal material. Microscopic identification is costly in practical research, as the instruments are expensive and require the identification worker to have expertise in plant anatomy, plant microsection preparation, and the operation of relevant microscopic instruments. Peucedanum praeruptorum contains a variety of complex chemical components, and identification based on a few physicochemical characteristics is sometimes insufficient. Furthermore, physicochemical identification usually requires complex pretreatment of the medicinal material, which is time-consuming and labor-intensive. For example, Huang Donglan et al. (Rapid Identification of Peucedanum praeruptorum and its adulterant Saposhnikovia divaricata by Second Derivative Infrared Spectroscopy. Journal of Shaoguan University [J]. 2009, 12) compared the infrared spectra of Peucedanum praeruptorum and Saposhnikovia divaricata using one-dimensional infrared spectroscopy. Although this method can achieve rapid identification of Peucedanum praeruptorum and Saposhnikovia divaricata, it requires highly skilled operators, and the test results are greatly limited by the environment and human subjective initiative. Therefore, traditional identification methods are difficult to use simply, quickly and accurately to effectively distinguish counterfeit Angelica dahurica products.

[0004] Hu Yijuan (Study on HPLC fingerprint spectrum of Angelica dahurica [J]. Chinese Journal of Traditional Chinese Medicine Science and Technology, 2012, 19(5): 437-439) conducted a fingerprint spectrum study on Angelica dahurica, but her HPLC detection method only obtained 6 common peaks, and a large number of peaks in the characteristic chromatogram separated in a short time, resulting in uneven baseline, which led to inaccurate analysis of the effective components in Angelica dahurica and affected the quality judgment. Chinese patent application CN110297045A disclosed a characteristic spectrum detection method for Angelica dahurica formula granules, which can simultaneously detect Angelica dahurica A and Angelica dahurica B, and can identify 8 characteristic peaks. It can be used for the quality detection of formula granules and reflects the overall appearance of the multi-component formula granules. However, the method has relatively few characteristic peaks, and the separation and symmetry of individual characteristic peaks (such as Angelica dahurica B) are poor. Moreover, the analysis time is long, so it cannot be applied to the rapid identification of Angelica dahurica and its counterfeits.

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

[0006] This invention addresses the problems existing in the prior art by providing a method and application for identifying fingerprint spectra of Peucedanum praeruptorum and its counterfeits. This method can effectively distinguish between genuine Peucedanum praeruptorum and counterfeits, and can identify counterfeits with an adulteration rate of more than 30%. Compared with traditional identification methods, it can more quickly, accurately and comprehensively distinguish counterfeits of Peucedanum praeruptorum.

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

[0008] On the one hand, the present invention provides a method for identifying the fingerprint spectrum of Peucedanum praeruptorum and its counterfeits, comprising the following steps:

[0009] S1. Preparation of reference solution: Take angelica coumarin II, angelica A, angelica B and angelica E respectively to prepare a mixed reference solution;

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

[0011] S3. Ultra-high performance liquid chromatography determination: Inject the mixed reference 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, gradient elution is 0-25 min, 40%→65% mobile phase A, 60%→35% mobile phase B.

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

[0013] Preferably, the chromatographic conditions described in step S3 further include: the chromatographic column is ACQUITY. BEH C18 column, ACQUITY Either the HSS T3 column or the Pheromon SuperLu C18-AQ column.

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

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

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

[0017] Preferably, the test sample in step S2 includes at least one of the following: Peucedanum praeruptorum, Peucedanum praeruptorum var. ... var. praeruptorum, and Peucedanum praeruptorum var. praeruptorum.

[0018] More preferably, the Peucedanum praeruptorum herbal medicine includes any one of Peucedanum praeruptorum raw material and Peucedanum praeruptorum processed slices; the hard Peucedanum praeruptorum herbal medicine includes any one of hard Peucedanum praeruptorum raw material and hard Peucedanum praeruptorum processed slices; the hairy Peucedanum praeruptorum herbal medicine includes any one of hairy Peucedanum praeruptorum raw material and hairy Peucedanum praeruptorum processed slices; the cloudy Peucedanum praeruptorum herbal medicine includes any one of cloudy Peucedanum praeruptorum raw material and cloudy Peucedanum praeruptorum processed slices.

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

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

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

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

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

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

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

[0026] Preferably, 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.

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

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

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

[0030] Specifically, the adulterated Peucedanum praeruptorum includes at least one of pure hard Peucedanum praeruptorum, pure hairy Peucedanum praeruptorum, and pure cloud Peucedanum praeruptorum; the adulterated Peucedanum praeruptorum is pure hard Peucedanum praeruptorum, pure hairy Peucedanum praeruptorum, or pure cloud Peucedanum praeruptorum mixed into pure Peucedanum praeruptorum, wherein the mass of the adulterated Peucedanum praeruptorum is greater than or equal to 30% and less than 100% of the mass of pure Peucedanum praeruptorum.

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

[0032] This invention constructs a reference fingerprint spectrum of genuine Peucedanum praeruptorum using ultra-high performance liquid chromatography (UPLC), which can effectively distinguish Peucedanum praeruptorum adulterants (hard Peucedanum praeruptorum, hairy Peucedanum praeruptorum, and cloud Peucedanum praeruptorum). The similarity results between the fingerprint spectra of adulterated products and the reference Peucedanum praeruptorum at different proportions show that the identification method of this invention can identify adulterated Peucedanum praeruptorum with an adulteration rate of over 30%. Furthermore, the identification method of this invention has better durability and produces more refined fingerprint spectra. Compared with traditional identification methods, it can more quickly, accurately, and comprehensively distinguish Peucedanum praeruptorum from its adulterants. Attached Figure Description

[0033] Figure 1 The above is the UPLC chromatogram of the mixed reference standard in Example 1, where peak 3 is angelica coumarin II, peak 5 is angelica A, peak 12 is angelica B, and peak 14 is angelica E.

[0034] Figure 2 The UPLC diagram of the Peucedanum praeruptorum (Q1) sample in Example 1 is shown. Peak 3 is Peucedanum praeruptorum coumarin II, peak 5 is Peucedanum praeruptorum A, peak 12 is Peucedanum praeruptorum B, and peak 14 is Peucedanum praeruptorum E.

[0035] Figure 3 The fingerprint spectrum superimposed on the Peucedanum praeruptorum and its standard fingerprint spectrum in Example 1;

[0036] Figure 4The fingerprint spectrum of *Eupatorium fortunei* superimposed in Example 1 and its standard fingerprint spectrum;

[0037] Figure 5 The superimposed fingerprint spectrum of Angelica pubescens and its standard fingerprint spectrum are shown in Example 1;

[0038] Figure 6 The superimposed fingerprint spectrum of Angelica dahurica and its standard fingerprint spectrum in Example 1;

[0039] Figure 7 This is a graph showing the effect of adulteration rate on the similarity of the fingerprint spectrum of Angelica dahurica in Example 1;

[0040] Figure 8 The results of the investigation of different brands of chromatographic columns in Example 1 are shown in the figure.

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

[0042] Figure 10 The graph shows the flow velocity results from Example 1.

[0043] Figure 11 The UPLC diagrams of the test sample (Q1) and the mixed reference sample in Comparative Example 1 are shown. Peak 1 is angelica coumarin II, peak 2 is angelica A, peak 3 is angelica B, and peak 4 is angelica E.

[0044] Figure 12 The UPLC diagrams of the test sample (Q1) and the mixed reference sample in Comparative Example 2 are shown. Peak 1 is angelica coumarin II, peak 2 is angelica A, peak 3 is angelica B, and peak 4 is angelica E. Detailed Implementation

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

[0046] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, which should be understood to include values ​​close to them. For numerical ranges, one or more new numerical ranges can be obtained by combining the endpoint values ​​of the ranges, the endpoint values ​​of the ranges with individual point values, and individual point values ​​with each other, and these numerical ranges should be considered as specifically disclosed herein. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein include both singular and plural indicators. Numerical ranges expressed by endpoints include all numerical values ​​and fractions within the corresponding range, as well as the expressed endpoints.

[0047] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort should fall within the scope of protection of this invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described, unless otherwise specified, are commercially available.

[0048] Experimental instruments and reagents

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

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

[0051] Experimental materials

[0052] The sample information used in the experiment is detailed in Table 1. The collection period was from January 2020 to October 2023. The samples were identified by Researcher Yu Jinbao of Jiangxi Provincial Academy of Traditional Chinese Medicine as follows: Q1-Q32 are white-flowered Peucedanum praeruptorum, Y1-Y12 are sparsely hairy Peucedanum praeruptorum, M1-M9 are short-sliced ​​Ligusticum striatum, and H1-H15 are red Peucedanum praeruptorum.

[0053] Table 1 Sample Information Table

[0054]

[0055]

[0056]

[0057] Example 1

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

[0059] 2. Solution preparation:

[0060] (1) Preparation of mixed reference solution

[0061] Accurately weigh 9.68 mg of angelica coumarin II, 11.60 mg of angelica azone, 11.34 mg of angelica ethylsin, and 10.38 mg of angelica ethephon E, and place them in four 25 mL volumetric flasks. Dilute to the mark with methanol and mix well to obtain a mixed reference stock solution. Accurately measure 1 mL of each of the four reference stock solutions into the same 10 mL volumetric flask to obtain mixed reference solutions with concentrations of 37.9, 46.1, 45.2, and 40.7 μg / mL, respectively.

[0062] (2) Preparation of the test solution

[0063] Take approximately 0.1 g of the powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of methanol, seal tightly, weigh it, sonicate (power 250 W, frequency 50 kHz) for 10 minutes, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it through a 0.22 μm microporous membrane, and collect the filtrate to obtain the product.

[0064] 3. Establishment of fingerprint patterns

[0065] Take angelica root (Q1-Q32), hard angelica root (Y1-Y12), hairy angelica root (M1-MQ09), and cloud angelica root (H1-H15) respectively, and prepare test solutions according to the above-described "Preparation Method of Test Solution". Inject and determine the chromatograms under the above-described "Chromatographic Conditions", and record the chromatograms (e.g., ...). Figure 1 and Figure 2 (As shown). The chromatographic data of Qianhu (Q1-Q20), Hard Qianhu (Y1-Y12), Hairy Qianhu (M1-MQ09), and Cloud Qianhu (H1-H15) were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)". Q1, Y1, M1, and H1 were set as reference spectra, respectively. The median method was selected as the standard chromatogram generation method, and the time window width was set to 0.1 min. After multi-point correction, full-spectrum peak matching was performed to obtain the standard chromatograms R and superimposed chromatograms of Qianhu, Hard Qianhu, Hairy Qianhu, and Cloud Qianhu (see...). Figures 3-6), and save the standard diagram R of Peucedanum praeruptorum.

[0066] result:

[0067] The peak matching results show that 16, 13, 17 and 12 common peaks were identified for Qianhu, hard Qianhu, hairy Qianhu and cloud Qianhu, respectively. Among them, peak No. 12 (white flower Qianhu ethyl) and peak No. 31 (unknown peak) have moderate peak areas and good separation. Therefore, peak No. 12 was selected as the reference peak for Qianhu, hard Qianhu and hairy Qianhu, and peak No. 31 was selected as the reference peak for cloud Qianhu. The similarity scores of 20 batches of *Peucedanum praeruptorum* (Qianhu) to its standard pattern 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 similarity scores of 12 batches of hard *Peucedanum praeruptorum* (Qianhu) to its standard pattern were: 0.980, 0.989, 0.953, 0.983, 0.976, 0.988, 0.988, 0.993, 0.993, 0.979, 0.9 The similarity scores of 9 batches of *Radix Angelicae Sinensis* and their standard chromatograms were 0.989, 0.917, 0.982, 0.974, 0.987, 0.986, 0.982, 0.974, and 0.920, respectively, all ≥0.917; the similarity scores of 15 batches of *Radix Angelicae Sinensis* and their standard chromatograms 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, and 0.991, respectively, all ≥0.986, indicating that the differences between the batches of samples were small.

[0068] By comparing with reference standards, four components were identified: angelica pubescens coumarin II (peak 3), angelica pubescens A (peak 5), angelica pubescens B (peak 12), and angelica pubescens E (peak 14). Nine peaks were shared between *Peucedanum praeruptorum* (hard angelica) and *Peucedanum praeruptorum* (hairy angelica) and *Peucedanum praeruptorum* (hairy angelica) and *Peucedanum praeruptorum* (hairy angelica) and *Peucedanum praeruptorum* (yellow angelica) and *Peucedanum praeruptorum* (yellow angelica) and *Peucedanum praeruptorum* (cloud ...

[0069] 4. Methodological Examination:

[0070] Take Peucedanum praeruptorum (Q1) and prepare a 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 perform 6 consecutive injections and determinations, recording the chromatograms. Among them, Peucedanum praeruptorum peak 12 (white flower peucedanin) is used as the reference peak. The RSD of the relative retention time of the common peaks is calculated to be less than 0.62%, and the RSD of the relative peak areas is less than 1.83%, indicating that the instrument precision is good.

[0071] Take the same batch of Peucedanum praeruptorum (Q1) and 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. The RSD of the relative retention time of the common peak is less than 0.56% and the RSD of the relative peak area is less than 1.24%, indicating that the method has good repeatability.

[0072] The Peucedanum praeruptorum 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, and then injected and measured under the "Chromatographic Conditions" in Example 1 above. The calculated RSD of the relative retention time of the common peak was less than 0.32%, and the RSD of the relative peak area was less than 1.66%, indicating that the test solution prepared by this method has good stability.

[0073] 5. Comparison of similarity among different types of Peucedanum praeruptorum herbs

[0074] According to the Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 version), with the working state selected as "Analysis and Testing", the standard chromatogram R of Peucedanum praeruptorum and the chromatographic data of Peucedanum praeruptorum (Q21-Q32), Peucedanum praeruptorum (Y01-Y12), Peucedanum praeruptorum (M01-MQ09) and Peucedanum praeruptorum (H01-H15) were imported respectively. The standard chromatogram of Peucedanum praeruptorum was used as the reference chromatogram. The median method was used, and the time window was selected as 0.1 min. After multi-point correction and automatic matching of full spectrum peaks, the similarity was calculated. The results are shown in Table 2.

[0075] Table 2. Similarity between different types of Peucedanum praeruptorum and the standard Peucedanum praeruptorum diagram.

[0076]

[0077] As shown in Table 2, the similarity between the 12 batches of Peucedanum praeruptorum samples and the standard Peucedanum praeruptorum pattern was high, ranging from 0.949 to 0.997, indicating that the fingerprint patterns of Peucedanum praeruptorum from different origins and cultivation methods were not significantly different. The similarity between the hard Peucedanum praeruptorum, hairy Peucedanum praeruptorum, and cloud Peucedanum praeruptorum and the standard Peucedanum praeruptorum pattern ranged from 0.070 to 0.240, indicating that the fingerprint patterns of counterfeit Peucedanum praeruptorum products and genuine Peucedanum praeruptorum products were significantly different, and the similarity could effectively distinguish Peucedanum praeruptorum products and their counterfeits.

[0078] 6. The effect of adulteration rate on the similarity of fingerprint spectra of Angelica dahurica

[0079] To investigate the effects of adulteration with hard, hairy, and cloud-like Peucedanum praeruptorum on fingerprint similarity, hard (Y1), hairy (M1), and cloud-like (H1) Peucedanum praeruptorum samples were added to Peucedanum praeruptorum (Q1) at mass percentages of 0%, 10%, 20%, 30%, 40%, 50%, 70%, 90%, and 100%, respectively. The similarity between the different adulterated Peucedanum praeruptorum samples and the standard Peucedanum praeruptorum fingerprint spectrum was calculated. The results are shown below. Figure 7 The results show that the similarity of the samples gradually decreases with the increase of the adulteration rate. When 30% of hard angelica, hairy angelica, and cloud angelica are added to angelica, 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 identify adulterated angelica products with an adulteration rate of more than 30%.

[0080] In summary, the fingerprint similarity test results indicate that when the similarity of the samples is <0.3, the samples are adulterated with Peucedanum praeruptorum; when 0.3 < similarity <0.9, the adulteration rate of the samples is ≥30%. Therefore, the constructed Peucedanum praeruptorum UPLC fingerprint spectrum can be used to identify common adulterants of Peucedanum praeruptorum and to determine Peucedanum praeruptorum adulterated with an adulteration rate of 30% or higher.

[0081] 7. Durability Test

[0082] (1) Chromatographic column investigation

[0083] Using Peucedanum praeruptorum (Q1) as the sample, and following the method described in Example 1 above, the chromatographic column ACQUITY was tested. BEHC18 column (1.7μm, 2.1mm × 100mm), ACQUITY The results were investigated using HSS T3 (1.8µm, 2.1×100mm) and Phiromens SuperLu C18-AQ (1.8µm, 2.1×100mm), and are shown in the table below. Figure 8 Tables 3 and 4.

[0084] Table 3. Column robustness study—relative retention time ratios

[0085]

[0086]

[0087] Table 4. Column robustness test—relative peak area ratio

[0088]

[0089] The results showed that when the samples were detected using the three chromatographic columns, the common peaks were relatively symmetrical and the resolution was good. The RSD of the relative retention time of the common peaks ranged from 0.26% to 6.07%, and the RSD of the relative peak area of ​​the common peaks ranged from 0.66% to 8.75%.

[0090] (2) Column temperature investigation

[0091] Using Peucedanum praeruptorum (Q1) as the sample, the column temperatures of 25℃, 30℃, and 35℃ were investigated according to the method described in Example 1 above. The results are shown below. Figure 9 Tables 5 and 6.

[0092] Table 5 Column Temperature Study—Ratio of Relative Retention Time

[0093]

[0094]

[0095] Table 6 Column Temperature Investigation—Relative Peak Area Ratio

[0096]

[0097] The results showed that when the column temperature was 25-35℃, the chromatogram peaks were relatively symmetrical, the resolution was good, and the RSD of the relative retention time of the common peaks was less than 3%, indicating good method robustness.

[0098] (3) Flow velocity investigation

[0099] Using Peucedanum praeruptorum (Q1) as the sample, the flow rates of 0.2 mL / min, 0.25 mL / min, and 0.3 mL / min were investigated according to the method described in Example 1 above. The results are shown below. Figure 10 Tables 7 and 8.

[0100] Table 7 Flow velocity assessment—Ratio of relative retention time

[0101]

[0102] Table 8. Flow velocity analysis—Ratio of relative peak area

[0103]

[0104] The results showed that when the flow rates were 0.2 ml / min, 0.25 ml / min, and 0.3 ml / min, the relative retention times (RSDs) of each common peak were 0.37-7.31%. At a flow rate of 0.25 ml / min, the chromatogram peak shape was better and the resolution was moderate.

[0105] Comparative Example 1

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

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

[0108] The results are as follows Figure 11 As shown, this method has relatively few characteristic peaks, and the separation and symmetry of individual characteristic peaks (white peucedanum ethyl) are poor. In addition, the analysis time is long (70 min), so it cannot be applied to the rapid identification of peucedanum and its counterfeits.

[0109] Comparative Example 2

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

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

[0112] The results are as follows Figure 12 As shown, this method has poor durability and cannot effectively separate the indicative components, angelica dahurica A and angelica dahurica E; moreover, it identifies few characteristic peaks and cannot accurately and effectively distinguish genuine angelica dahurica from counterfeit angelica dahurica.

[0113] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for identifying the fingerprint spectrum of Peucedanum praeruptorum and its counterfeits, characterized in that: Includes the following steps: S1. Preparation of reference solution: Take angelica coumarin II, angelica A, angelica B and angelica E respectively to prepare a mixed reference solution; S2. Preparation of the test solution: Take the test sample, extract it with methanol, and then filter it to obtain the test solution; S3. Ultra-high performance liquid chromatography determination: Inject the mixed reference 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, gradient elution is 0-25 min, 40%→65% mobile phase A, 60%→35% mobile phase B. S4. Establishment of fingerprint spectrum: Record the results of ultra-high performance liquid chromatography and establish fingerprint spectrum; The mass-to-volume ratio of the test sample to methanol in step S2 is 0.1-0.15 g: 10 mL; The chromatographic conditions described in step S3 further include: the chromatographic column is ACQUITY UPLC. ® BEH C18 column, ACQUITY UPLC ® Any one of the following: HSS T3 column and Phiromon SuperLu C18-AQ column; The chromatographic conditions described in step S3 also include: column temperature of 30°C; flow rate of 0.25 mL / min; and wavelength of 321 nm.

2. The identification method according to claim 1, characterized in that: The chromatographic column is an ACQUITY UPLC. ® BEHC18 chromatographic column.

3. The identification method according to claim 1, characterized in that: The test sample mentioned in step S2 includes at least one of the following: Peucedanum praeruptorum, Peucedanum praeruptorum var. ...

4. The identification method according to claim 3, characterized in that: The term "Peucedanum praeruptorum" includes any one of the following: raw Peucedanum praeruptorum material and processed Peucedanum praeruptorum slices; "Hard Peucedanum praeruptorum" includes any one of the following: raw Peucedanum praeruptorum material and processed Peucedanum praeruptorum slices; "Hairy Peucedanum praeruptorum" includes any one of the following: raw Peucedanum praeruptorum material and processed Peucedanum praeruptorum slices; "Cloudy Peucedanum praeruptorum" includes any one of the following: raw Peucedanum praeruptorum material and processed Cloudy Peucedanum praeruptorum slices.

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

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

7. The identification method according to claim 6, characterized in that: The conditions for ultrasonic extraction include: power 250W, frequency 50kHz, and time 10 minutes.

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

9. 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.

10. The identification method according to claim 9, characterized in that: The volume of the mixed reference solution is 2 μl, and the volume of the test solution is 2 μl.

11. The application of the identification method according to any one of claims 1-10 in distinguishing between Peucedanum praeruptorum and its counterfeit products.

12. The application according to claim 11, characterized in that: The adulterated products of Qianhu include mixed adulterated products and adulterated products.

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