Construction method of characteristic chromatograms of common carpesium fruit and preparation thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, and particularly provides a construction method of specific chromatograms of common carpesium fruit and a preparation thereof. Comprising the following steps: by taking octadecylsilane chemically bonded silica as a filling agent and a mobile phase comprising methanol and an acid-containing aqueous solution, carrying out gradient elution, and screening through a large number of experiments to obtain a specific elution procedure, so as to obtain 13 or more common characteristic peaks, and realizing the purification of the chlorogenic acid, the caffeic acid, the chlorogenic acid, the cryptochlorogenic acid, the 1, 4-dicaffeoylquinic acid, the 1, 4-dicaffeoylquinic acid, the 1, 4-dicaffeoylquinic acid, the 1, 4-dicaffeoylquinic acid and the 1, 4-dicaffeoylquinic acid. The method can effectively separate characteristic peaks of 2, 5-dicaffeoylquinic acid, cosmosiin and carabrone, is good in peak shape, stable in base line, short in detection time, provides a basis for quality detection and control of the common carpesium fruit and the preparation thereof, can fully reflect the integrity and characteristics of the common carpesium fruit and the preparation thereof, is simple to operate, and is suitable for industrial production. The method is suitable for quality control of different preparation types of common carpesium fruit.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for constructing a characteristic spectrum of a louse and a preparation thereof. Background Art

[0002] Carpesium abrotanoides L. is a dried mature fruit of the Asteraceae plant. The fruit is harvested in autumn when it is ripe, dried in the sun, and impurities are removed. It is mainly used for ascariasis, enterobiasis, tapeworm disease, abdominal pain caused by worm accumulation, and malnutrition in children. According to literature reports, the main chemical components of Carpesium abrotanoides are sesquiterpenoids (Carpesium abrotanoides L., etc.), fatty acids, and organic acid compounds (such as chlorogenic acid, etc.). Carpesium abrotanoides standard decoction is obtained by extracting, concentrating, and drying the traditional Chinese medicine Carpesium abrotanoides slices.

[0003] The quality control of crane lice in the 2020 edition of the Chinese Pharmacopoeia includes items such as original plant varieties, preparation of medicinal pieces, properties of medicinal pieces, and physical and chemical identification, but there are no items related to content determination. Chinese patent CN116754688A discloses a detection method for crane lice and its quality control method, using octadecylsilane bonded silica gel as a filler; methanol as mobile phase A, and 0.2% phosphoric acid aqueous solution as mobile phase B, eluted according to a specific gradient elution procedure, and the resulting spectrum contains 8 characteristic peaks. There are fewer characteristic peaks in this patent, and the detection time is at least 95 minutes, which is time-consuming and cannot comprehensively and holistically detect and control the quality of crane lice and its preparations. Summary of the invention

[0004] The object of the present invention is to provide a method for constructing a characteristic spectrum of lice and a preparation thereof. The characteristic spectrum constructed by the method of the present invention has at least 13 characteristic peaks and good peak separation. The characteristic spectrum is used for quality detection to improve the accuracy of the analysis result.

[0005] To this end, the present invention discloses a method for constructing a characteristic spectrum of a louse and a preparation thereof, comprising the following steps:

[0006] (1) Preparation of test solution;

[0007] (2) The sample solution is tested by ultra-high performance liquid chromatography, the filler is octadecylsilane bonded silica gel, and the mobile phase includes methanol and acid-containing aqueous solution;

[0008] Gradient elution, the gradient elution program includes: 0→12 minutes→18 minutes→20 minutes→25 minutes→28 minutes→34 minutes→42 minutes, and the volume percentage of methanol in the mobile phase is 7%→18%→30%→35%→40%→45%→47%→60%.

[0009] Further, step (1) comprises:

[0010] 1) Take the object to be tested, add a solvent to extract it, and obtain an extract;

[0011] 2) Separate the solid and liquid of the extract and take the liquid, which is the test solution;

[0012] Preferably, step (1) also satisfies any one or more of the following AE:

[0013] A. In step 1), the solvent is selected from at least one of water, methanol and ethanol;

[0014] B. In step 1), the ratio of the mass of the analyte to the volume of the solvent is 0.1-2 g:10-250 mL;

[0015] C. In step 1), the extraction method is reflux extraction or ultrasonic extraction, and the extraction time is 15min-120min;

[0016] D. In step 2), the solid-liquid separation is centrifugation or filtration;

[0017] E. In step 1), the analyte is selected from one or more of the following: Chinese lice medicinal materials, Chinese lice decoction pieces, Chinese lice granules, Chinese lice decoctions, and Chinese lice powders.

[0018] Furthermore, the acid-containing aqueous solution is selected from at least one of a formic acid aqueous solution and a phosphoric acid aqueous solution; preferably, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%; preferably, the volume percentage of formic acid in the formic acid aqueous solution is 0.05-0.1%.

[0019] Furthermore, the chromatographic conditions of the ultra-high performance liquid chromatography in step (2) also include: a detection wavelength of 210-280 nm, a flow rate of 0.23-0.27 ml / min, a column temperature of 25-33° C., a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6-1.8 μm; preferably, the detection wavelength is 240 nm; preferably, the column temperature is 30° C.; preferably, the injection volume is 2 μl.

[0020] Furthermore, the gradient elution program also includes, 42 minutes→43 minutes, the volume percentage of methanol in the mobile phase is 60%→7%, or the gradient elution program also includes, 42 minutes→43 minutes→48 minutes, the volume percentage of methanol in the mobile phase is 60%→7%→7%.

[0021] Furthermore, the construction method also includes the step of preparing a reference solution using at least one reference substance selected from the group consisting of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmos glycoside, and asparagus lactone, and the step of detecting the reference solution by ultra-high performance liquid chromatography according to any of the construction methods to obtain a reference spectrum of the reference substance.

[0022] Furthermore, the characteristic spectrum of the crane louse and its preparation obtained by the construction method is selected from any one of the following (1)-(2):

[0023] (1) The characteristic spectrum of the louse and its preparation has 13 characteristic peaks, with peak 9 as the reference peak, and the relative retention times of characteristic peaks 1-8 and peaks 10-13 and the reference peak are within the range of ±10% of the specified values; the specified values ​​are: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), 1.39 (peak 13);

[0024] (2) The characteristic spectrum of the described sphaerocephala and its preparation has 13 characteristic peaks, the peak corresponding to the peak of the 1,5-dicaffeoylquinic acid reference substance is the reference peak, and the relative retention time of the characteristic peaks 1-8 and peaks 10-13 and the reference peak is within the range of ±10% of the specified value; the specified values ​​are: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), and 1.39 (peak 13).

[0025] The invention also provides application of the construction method in quality control of lice and preparations thereof.

[0026] The present invention also provides a quality control method for crane lice and its preparation, comprising the step of comparing the characteristic spectrum of the crane lice product to be tested with the characteristic spectrum of the crane lice and its preparation; the characteristic spectrum of the crane lice product to be tested is obtained by using the crane lice product to be tested according to the above-mentioned construction method, and the characteristic spectrum of the crane lice and its preparation is the characteristic spectrum of the above-mentioned crane lice and its preparation.

[0027] The present invention also provides a method for distinguishing the lice and its preparation from counterfeits, comprising:

[0028] Use the product to be identified as a test sample and construct a characteristic spectrum of the product to be identified according to the above construction method;

[0029] Compare the obtained characteristic spectrum of the product to be identified with the characteristic spectrum constructed by the above construction method, and perform identification according to the comparison result;

[0030] The counterfeit products are southern crane louse and / or southern crane louse preparations.

[0031] The technical solution of the present invention has the following advantages:

[0032] 1. The method for constructing the characteristic spectrum of the crane louse and its preparation provided by the present invention uses octadecylsilane bonded silica gel as a filler, and the mobile phase includes methanol and an acid-containing aqueous solution, and gradient elution is performed. A specific elution program is obtained through a large number of experimental screenings, and 13 or more common characteristic peaks are obtained, and effective separation of characteristic peaks including neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmosin, and tianmingjing lactone is achieved. The peak shape is good, the baseline is stable, the measured components can be completely separated, and the detection time is short, which provides a basis for the quality inspection and control of the crane louse and its preparation, can fully reflect the integrity and characteristic of the crane louse and its preparation, and the method is simple to operate and can be applicable to the quality control of different types of crane louse preparations.

[0033] 2. The method for constructing the characteristic spectrum of the crane louse and its preparation provided by the present invention, after identification by the reference substance, peaks 3, 4, 5, 6, 8, 9, 10, and 12 are respectively neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmos glycoside, and tianmingjing lactone, which are well separated and have moderate areas. The accuracy of crane louse quality control can be achieved in a qualitative manner by using one or more of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmos glycoside, and tianmingjing lactone as reference. The 13 or more characteristic peaks determined in the characteristic spectrum can well guarantee the accuracy of the method in the quality control of crane louse.

[0034] 3. The quality control method of the lice and its preparation provided by the present invention comprises the step of comparing the characteristic spectrum of the lice product to be tested with the characteristic spectrum of the lice and its preparation, which can control the material transfer of the index components in the lice medicinal materials, lice decoction pieces, lice granules, lice decoctions and lice powders, and can also control the stability of the quality of the characteristic components of the lice medicinal materials, lice decoction pieces, lice granules, lice decoctions and lice powders as a whole. The method is simple to operate, has high precision, good stability, good repeatability, high accuracy and fast analysis rate.

[0035] 4. The method for distinguishing the crane louse and its preparations from counterfeits provided by the present invention has strong specificity and can quickly and accurately distinguish and identify the crane louse and its pharmaceutical preparations, the southern crane louse and its pharmaceutical preparations and their mixed counterfeits. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 It is the chromatogram overlay of the test solution and the reference solution in Example 1.

[0038] Figure 2 It is the chromatogram of the control medicinal material reference solution in Example 1.

[0039] Figure 3 It is the reference characteristic spectrum in Example 2.

[0040] Figure 4 It is an overlay of the chromatograms of the test solution in Example 3 - freeze-dried powder of standard decoction of crane lice and freeze-dried powder of standard decoction of southern crane lice.

[0041] Figure 5 It is the chromatogram of the test solution in Example 4.

[0042] Figure 6 It is the chromatogram of the test solution in Comparative Example 1.

[0043] Figure 7 It is the chromatogram of the test solution in Comparative Example 2.

[0044] Figure 8 It is the chromatogram of the test solution in Comparative Example 3.

[0045] Fig. 9 This is the chromatographic overlay of the test solution in Experimental Example 1 - an investigation of the type and concentration of the mobile phase.

[0046] Fig.10 It is the chromatogram overlay of the test solution in Experimental Example 1 - the choice of wavelength.

[0047] Fig.11 It is the chromatogram overlay of the test solution in Experimental Example 1 - the choice of column temperature.

[0048] Fig.12 This is the chromatogram of the test solution in Experimental Example 1. The chromatographic column selected is Waters CORTECS T3.

[0049] Fig.13 This is the chromatogram of the test solution in Experimental Example 1. The chromatographic column selected is YMC aq-C18.

[0050] Fig.14 This is the chromatogram of the negative control solution in Experimental Example 3 - Specificity test.

[0051] Fig.15 This is the chromatogram of the test solution in Experimental Example 3 - Specificity test.

[0052] Figure symbols: Peak 3: neochlorogenic acid; Peak 4: caffeic acid; Peak 5: chlorogenic acid; Peak 6: cryptochlorogenic acid; Peak 8: 1,4-dicaffeoylquinic acid; Peak 9 (S): 1,5-dicaffeoylquinic acid; Peak 10: cosmoside; Peak 12: tianmingjing lactone. DETAILED DESCRIPTION

[0053] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0054] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0055] The instruments and reagents used in the present invention are as follows:

[0056] 1. Chromatography: (1) Waters ACQUITY UPLC I-Class chromatography system, including quaternary solvent manager (ACQ-QSM), automatic sample injector (ACQ-FTN), original imported chromatography column oven (ACQ-CM), diode array ultraviolet detector (ACQ-TUV), and Empower chromatography management system.

[0057] 2. Electronic analytical balance: Mettler Toledo NewClassic MS one hundred thousandth balance, Jing TianFA2044A one ten thousandth balance.

[0058] 3. Chromatographic columns: (1) Waters CORTECS T3, 100 mm × 2.1 mm, 1.6 μm; (2) Agilent Eclipse Plus C18 RRHD, 100 mm × 2.1 mm, 1.8 μm; (3) Chromatographic column YMC aq-C18, 100 mm × 2.1 mm, 1.9 μm.

[0059] 4. Reagents: methanol (chromatographic grade), water (ultrapure water), formic acid (analytical grade).

[0060] 5. Test drugs: Caffeic acid (110885-201703, 99.7%) and chlorogenic acid (110753-202119, 96.3%) were purchased from China Food and Drug Inspection Institute; neochlorogenic acid (DSTDX001503, 98%) and cryptochlorogenic acid (DST210427-035, 98%) were purchased from Lemeitian Pharmaceutical Co., Ltd.; 1,4-dicaffeoylquinic acid (050184-202309, 98%), cosmosin (170088-202106, 98%), and tianmingjing lactone (200162-202311, 98%) were purchased from Shanghai Hongyong Biotechnology Co., Ltd.; 1,5-dicaffeoylquinic acid (000322-202309, 98%) was purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd.

[0061] The control medicinal material of Psoralea corylifolia (121131-202102) was purchased from China Food and Drug Inspection Institute.

[0062] Preparation method of freeze-dried powder of standard decoction of crane lice: take crane lice decoction pieces, heat reflux extraction twice, add water with a weight of 8 times the weight of the decoction pieces for the first time, soak for 30 minutes, heat reflux extraction for 30 minutes, filter, add water with a weight of 6 times the weight of the decoction pieces for the second time, extract for 25 minutes, filter, combine the filtrates after the two filtrations, concentrate the filtrate to a relative density of 1.05g / mL (detected at 60°C), and freeze-dry the concentrate to obtain the freeze-dried powder of the standard decoction.

[0063] According to the preparation method of the freeze-dried powder of the standard decoction of crane lice, the crane lice were replaced with the southern crane lice to prepare the freeze-dried powder of the standard decoction of southern crane lice.

[0064] Example 1

[0065] This embodiment provides a method for constructing a characteristic spectrum of a louse and its preparation, comprising the following steps:

[0066] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.6 μm); methanol was used as mobile phase A, 0.2% phosphoric acid aqueous solution (V:V) was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1 below; the flow rate was 0.25 ml per minute, the column temperature was 30°C, and the detection wavelength was 240 nm. The number of theoretical plates was calculated based on 1,5-dicaffeoylquinic acid and should not be less than 5000.

[0067] Table 1 Elution gradient

[0068]

[0069]

[0070] Preparation of reference solution: Take 1 g of the control medicinal material of Glechoma longituba, add 50 ml of water, heat and reflux for 60 minutes, filter, evaporate the filtrate to dryness, add 25 ml of 50% methanol aqueous solution (V:V) to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the control medicinal material. Take appropriate amounts of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmos glycoside, and tianmingjing lactone reference substances, accurately weigh them, and use 50% methanol aqueous solution (V:V) to prepare a solution containing 0.2 mg of neochlorogenic acid, 0.1 mg of caffeic acid, 0.1 mg of chlorogenic acid, 0.1 mg of cryptochlorogenic acid, 0.1 mg of 1,4-dicaffeoylquinic acid, 0.2 mg of 1,5-dicaffeoylquinic acid, 0.1 mg of cosmos glycoside, and 0.1 mg of tianmingjing lactone per 1 ml as a mixed solution of reference substances.

[0071] Take appropriate amounts of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmos glycoside, and tianmingjing lactone reference substances, accurately weigh them, and use 50% methanol aqueous solution (V:V) to prepare individual reference substance solutions containing 0.2 mg of neochlorogenic acid, 0.1 mg of caffeic acid, 0.1 mg of chlorogenic acid, 0.1 mg of cryptochlorogenic acid, 0.1 mg of 1,4-dicaffeoylquinic acid, 0.2 mg of 1,5-dicaffeoylquinic acid, 0.1 mg of cosmos glycoside, and 0.1 mg of tianmingjing lactone per 1 ml.

[0072] Preparation of test solution: Take an appropriate amount of freeze-dried powder of standard decoction of crane lice, grind it into powder, take about 0.1g, put it in a stoppered conical flask, add 25ml of 50% methanol aqueous solution (V:V), ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0073] The determination method is to accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0074] Test results see Figure 1-2, Table 2, there are 13 characteristic peaks in the chromatogram of the test solution, and the retention times should correspond to the 13 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 3, 4, 5, 6, 8, 9, 10, and 12 should correspond to the retention times of the peaks of the reference material of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmosin, and tianmingjing lactone. The peak corresponding to the peak of 1,5-dicaffeoylquinic acid reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value. The specified value is: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), and 1.39 (peak 13).

[0075] Table 2 System adaptability parameters

[0076]

[0077] Example 2

[0078] 18 batches of freeze-dried powder of standard decoction of lice were taken and characteristic spectra of 18 batches of freeze-dried powder of standard decoction of lice were obtained according to the method of Example 1. The results are shown in Table 3-4.

[0079] Table 3 Results of relative retention time determination of characteristic spectra of 18 batches of standard decoction of Helix sutchuenensis slices (lyophilized powder)

[0080]

[0081]

[0082]

[0083] Table 4 Results of relative peak area determination of characteristic spectra of 18 batches of standard decoction of Heshi decoction pieces (lyophilized powder)

[0084]

[0085]

[0086] The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate the reference characteristic spectrum, such as Figure 3 , as shown in Table 5-6. Figure 3The control characteristic spectrum shown can analyze and compare the test results of the characteristic spectrum of the freeze-dried powder of the standard decoction, and is used for the quality control of the standard decoction. The characteristic peaks are identified and pointed out by UPLC and LC / MS / MS, and 13 chromatographic peaks with good separation in the characteristic spectrum are confirmed. According to the results of peak identification and peak positioning in the spectrum of the reference solution, it is confirmed that peak 3 in the characteristic spectrum is neochlorogenic acid, peak 4 is caffeic acid, peak 5 is chlorogenic acid, peak 6 is cryptochlorogenic acid, peak 8 is 1,4-dicaffeoylquinic acid, peak 9 is 1,5-dicaffeoylquinic acid, peak 10 is cosmosin, and peak 12 is tianmingjing lactone. The retention time of the chromatographic peak of 1,5-dicaffeoylquinic acid is in the middle of the chromatogram, the peak response is large, and the reference is easy to obtain. Therefore, 1,5-dicaffeoylquinic acid is taken as the S peak of the characteristic spectrum, and the relative retention time of each characteristic peak is calculated. The relative retention time of each characteristic peak is within ±10% of the specified value, and the specified value is: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), and 1.39 (peak 13).

[0087] The characteristic spectra of 18 batches of freeze-dried powder of standard decoction of Helix sutchuenensis were compared with the characteristic spectra of the control, and the similarity was between 0.969-1. The results are shown in Table 7.

[0088] Table 5 Relative retention time of common patterns of standard decoction of lice

[0089]

[0090] Table 6 Common pattern matching data of standard decoction of lice

[0091]

[0092] Table 7 Similarity results of characteristic spectra of 18 batches of standard decoctions of Heshi

[0093]

[0094]

[0095] Example 3

[0096] This embodiment provides a method for distinguishing lice and its preparations from counterfeits, comprising the following steps:

[0097] The test solution of louse was prepared according to the preparation method of the test solution in Example 1.

[0098] The standard decoction freeze-dried powder of the crane lice was replaced with the standard decoction freeze-dried powder of the southern crane lice, and three batches of southern crane lice test solutions were prepared according to the preparation method of the test solution in Example 1.

[0099] Take the test solution of crane louse and the test solution of southern crane louse for determination according to the liquid phase method in Example 1.

[0100] Test results see Figure 4 The characteristic spectrum results showed that the 13 characteristic peaks of the crane lice did not exist in the chromatograms of the three batches of the test solutions of the crane lice. The identification method of this embodiment can distinguish the two, which is conducive to accurate medication.

[0101] Example 4

[0102] This embodiment provides a method for constructing a characteristic spectrum of a louse and its preparation. The construction method is basically the same as the construction method of Example 1, and the only difference is the gradient elution program in the chromatographic conditions. The gradient elution program of this embodiment is shown in Table 8. Figure 5 ,Table 9. From the results in the chart, it can be seen that the 13 characteristic peaks in the chromatogram have good separation effect, good peak shape and stable baseline.

[0103] Table 8 Gradient elution program

[0104]

[0105] Table 9 Test results

[0106]

[0107]

[0108] Comparative Example 1

[0109] This comparative example provides a method for constructing a characteristic spectrum of the crane lice and its preparation. The test solution prepared in Example 1 was tested. The chromatographic conditions were as follows: the chromatographic column was Waters CORTECS T3 (100mm×2.1mm, 1.6μm), the column temperature was 30℃, the flow rate was 0.3ml per minute, the injection volume was 1μl, and the gradient elution program was shown in Table 10. The test results are shown in Figure 6 From the results in the chart, we can see that the chromatographic peaks are concentrated at 22-26 minutes in the chromatogram, and some chromatographic peaks are not effectively separated, so the mobile phase elution gradient is further optimized.

[0110] Table 10 Gradient elution program

[0111]

[0112] Comparative Example 2

[0113] This comparative example provides a method for constructing a characteristic spectrum of the crane lice and its preparation. The test solution prepared in Example 1 was tested. The chromatographic conditions were as follows: the chromatographic column was Waters CORTECS T3 (100mm×2.1mm, 1.6μm), the column temperature was 30℃, the flow rate was 0.3ml per minute, the injection volume was 1μl, and the gradient elution program was shown in Table 11. The test results are shown in Figure 7 From the results in the chart, it can be seen that although the method of this comparative example advances the 22-26 minute chromatographic peak and further improves the separation effect of the chromatographic peak, the peak separation effect is still poor in the chromatogram, so the mobile phase elution gradient is further optimized.

[0114] Table 11 Gradient elution program

[0115]

[0116] Comparative Example 3

[0117] This comparative example provides a method for constructing a characteristic spectrum of the crane lice and its preparation. The test solution prepared in Example 1 was tested. The chromatographic conditions were as follows: the chromatographic column was Waters CORTECS T3 (100mm×2.1mm, 1.6μm), the column temperature was 30℃, the flow rate was 0.3ml per minute, the injection volume was 1μl, and the gradient elution program was shown in Table 12. The test results are shown in Figure 8 From the results in the chart, it can be seen that although the method of this comparative example further improves the separation effect of the middle chromatographic peak, the separation of the front 2-4 minute chromatographic peak is still poor.

[0118] Table 12 Gradient elution program

[0119]

[0120] Experimental Example 1 Establishment of Liquid Chromatography Conditions

[0121] 1. Investigation of mobile phase type and concentration

[0122] The chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.6 μm); methanol as the mobile phase A, 0.05% formic acid aqueous solution, 0.1% formic acid aqueous solution, 0.2% phosphoric acid aqueous solution, 0.4% phosphoric acid aqueous solution (V:V) as the mobile phase B, gradient elution was performed according to the provisions in Table 1; the flow rate was 0.25 ml per minute, the column temperature was 30° C., and the detection wavelength was 240 nm.

[0123] The test solution was prepared according to the preparation method of the test solution in Example 1, and the detection was carried out according to the determination method in Example 1 according to the chromatographic conditions under the above-mentioned "Investigation of mobile phase type and concentration".

[0124] Test results see Fig. 9 From the results, it can be seen that when formic acid is compared with phosphoric acid, phosphoric acid has a stable baseline, symmetrical peaks and good separation when used as the aqueous phase for elution. When different concentrations of phosphoric acid are compared, the separation effect of each characteristic peak is better when 0.2% phosphoric acid aqueous solution is used as the mobile phase, so methanol-0.2% phosphoric acid aqueous solution is preferred as the mobile phase.

[0125] 2. Selection of detection wavelength

[0126] Based on the above determination of the type and concentration of the mobile phase, the full wavelength detection method was used to observe the effect of the detection wavelengths of 210 nm, 240 nm, 260 nm, and 280 nm on the chromatographic peak response of the test solution. The full wavelength detection method was used to observe the effect of the chromatographic peak response.

[0127] Test results see Fig.10 As can be seen from the figure, compared with 210nm, 260nm, and 280nm, the chromatogram corresponding to the detection wavelength of 240nm has a stable baseline and high response, and 240nm is comprehensively selected as the detection wavelength of the characteristic spectrum.

[0128] 3. Selection of column temperature

[0129] Based on the above determination of the type and concentration of mobile phase and detection wavelength, the test solution was tested at column temperatures of 25°C and 30°C, respectively. The other liquid phase conditions were consistent with the methods under "Investigation of mobile phase type and concentration".

[0130] Test results see Fig.11 As can be seen from the figure, the separation of the chromatographic peaks is better at a column temperature of 30°C, so 30°C is preferably used as the column temperature for the characteristic spectrum of the crane louse.

[0131] 4. Selection of chromatographic columns

[0132] Based on the above determination of the type and concentration of mobile phase, detection wavelength, and column temperature, the test solution was tested on chromatographic columns Waters CORTECS T3 and YMC aq-C18, and the other liquid phase conditions were consistent with the methods under "Investigation of mobile phase type and concentration".

[0133] Test results see Figure 12-13 As can be seen from the figure, the separation of the chromatographic peaks under the Waters CORTECS T3 chromatographic column is good, so Waters CORTECS T3 is preferred as the chromatographic column for the characteristic spectrum of the crane louse.

[0134] 5. Determination of chromatographic conditions

[0135] According to the above optimization results, the chromatographic conditions of the characteristic spectrum are determined as follows:

[0136] Octadecylsilane bonded silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.6 μm); methanol was used as mobile phase A, 0.2% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 13 below; the flow rate was 0.25 ml per minute, the column temperature was 30°C, and the detection wavelength was 240 nm.

[0137] Table 13 Gradient elution program

[0138]

[0139] Experimental Example 2 Establishment of the preparation method of the test solution

[0140] Based on the chromatographic conditions determined under "Establishment of liquid chromatography conditions" in Experimental Example 1, the preparation method of the test solution was further investigated.

[0141] The prepared test solution was tested according to the chromatographic conditions determined under the "Establishment of Liquid Chromatographic Conditions" item of Experimental Example 1.

[0142] 1. Choice of treatment method

[0143] The effects of different extraction methods on the extraction efficiency of freeze-dried powder of standard decoction of Helicoverpa sutchuenensis were investigated, with the information content of chromatographic peaks and system suitability as the main evaluation indicators.

[0144] Extraction method: Take about 0.1g of freeze-dried powder of standard decoction of crane lice, add 25ml of 70% methanol aqueous solution (V:V), ultrasonically treat (power 300W, frequency 40kHz), heat reflux extraction for 30 minutes each, take out, cool, weigh again, make up the lost weight with 70% methanol aqueous solution, shake well, filter, and take the filtrate to obtain.

[0145] The test results are shown in Table 14. From the results, it can be seen that the system adaptability parameters of the chromatographic peaks of the test sample solutions obtained by the two extraction methods are relatively good, the separation and symmetry are good, and there is no obvious difference in the extraction between different extraction methods. Considering the simplicity of operation, ultrasonic treatment is preferred as the extraction method of the test sample.

[0146] Table 14 Comparison of extraction method-chromatographic peak system adaptability parameters

[0147]

[0148] 2. Selection of extraction solvent

[0149] On the basis of the above-determined treatment methods, water, ethanol, 30% ethanol aqueous solution, 50% ethanol aqueous solution, 70% ethanol aqueous solution, methanol, 30% methanol aqueous solution, 50% methanol aqueous solution, and 70% methanol aqueous solution (V:V) were selected as solvents to prepare the test solution.

[0150] The test results are shown in Table 15. From the results, it can be seen that compared with ethanol, 30% ethanol aqueous solution, 50% ethanol aqueous solution, and 70% ethanol aqueous solution, pure water, 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, and methanol can extract the chromatographic peaks better, among which 50% methanol aqueous solution has better peak shape, relatively better system adaptability parameters, and larger characteristic peak response, so 50% methanol aqueous solution is preferred as the extraction solvent.

[0151] Table 15 Comparison of extraction solvent-chromatographic peak system adaptability parameters

[0152]

[0153]

[0154]

[0155]

[0156] 3. Investigation of extraction time

[0157] Based on the treatment method and extraction solvent determined above, 15, 30, 45, and 60 minutes were selected as extraction times to prepare the test solution.

[0158] The test results are shown in Table 16. From the results, it can be seen that compared with the ultrasonic time of 15 min, when the ultrasonic time is 30 to 60 minutes, the peak area is higher, and the difference between the two is not large, so 30 minutes is preferably used as the ultrasonic time.

[0159] Table 16 Comparison of extraction time-chromatographic peak system adaptability parameters

[0160]

[0161]

[0162] 4. Investigation of solvent dosage

[0163] Based on the above-determined treatment method, extraction solvent, and extraction time, 10 ml, 25 ml, and 50 ml were selected as the dosage of 50% methanol aqueous solution to prepare the test solution.

[0164] The test results are shown in Table 17. From the results, it can be seen that when the solvent dosage is 10-50 ml, the peak area decreases proportionally, indicating that complete extraction can be achieved at a solvent dosage of 10-50 ml. Considering the chromatographic peak response, 25 ml is preferably used as the extraction solvent dosage.

[0165] Table 17 Comparison of solvent usage-chromatographic peak system adaptability parameters

[0166]

[0167]

[0168] 5. Selection of injection volume

[0169] Based on the above-determined treatment method, extraction solvent, extraction time, and solvent dosage, 1 μl, 2 μl, and 3 μl were selected as the injection volume to detect the test solution.

[0170] The test results are shown in Table 18. From the results, it can be seen that compared with the injection volume of 1 μl and 3 μl, the separation of the chromatographic peaks corresponding to the injection volume of 2 μl is better, and the peak height and peak width are relatively moderate, so the preferred injection volume is 2 μl as the injection volume.

[0171] Table 18 Comparison of injection volume-chromatographic peak system adaptability parameters

[0172]

[0173]

[0174] 6. Determination of the preparation method of the test solution

[0175] According to the above optimization results, the preparation method of the test solution was determined as follows: take about 0.1 g of freeze-dried powder of the standard decoction of crane lice, place it in a stoppered conical flask, add 25 ml of 50% methanol aqueous solution, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate; determine the injection volume to be 2 μl.

[0176] Experimental Example 3 Methodological Verification

[0177] 1. Precision test

[0178] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test solution was repeatedly injected 6 times for detection according to the chromatographic conditions in Example 1, and the retention time was recorded and the relative retention time (t / ts) of the common peak was calculated.

[0179] Peak 9 (1,5-dicaffeoylquinic acid) was taken as the S peak, and the relative retention times of peaks 1-8 and 10-13 were calculated.

[0180] The results are shown in Table 19 below. From the results, it can be seen that the RSD of the relative retention time of each characteristic peak and the reference S peak is less than 2%, indicating that the instrument precision is good.

[0181] Table 19 Instrument precision relative retention time test results

[0182]

[0183]

[0184] 2. Method repeatability test

[0185] Take the same batch number of lyophilized powder of standard decoction of schi, and prepare 6 test solutions according to the preparation method of the test solution in Example 1. The test is carried out according to the liquid phase conditions in Example 1, and the retention time is recorded and the relative retention time (t / ts) of the common peak is calculated.

[0186] The results are shown in Table 20. From the results, it can be seen that the RSD of the relative retention time of each characteristic peak and the reference S peak is less than 2%, indicating that the method has good repeatability.

[0187] Table 20 Method repeatability relative retention time test results

[0188]

[0189]

[0190] 3. Intermediate precision test (different operators)

[0191] Three inspectors, at different times, took the same batch of lyophilized powder of standard decoction of crane lice to prepare test solutions according to the preparation method of the test solution in Example 1, and used the same equipment to measure the retention time of the prepared test solutions and calculate the relative retention time (t / ts) of the common peaks.

[0192] The results are shown in Table 21. From the results, it can be seen that the RSD of the relative retention time of each characteristic peak and the reference S peak is less than 2%, indicating that the intermediate precision of this method is good.

[0193] Table 21 Intermediate precision relative retention time test results (different operators)

[0194]

[0195] 4. Specificity test

[0196] Select blank solvent as negative control solution (50% methanol aqueous solution), prepare test solution according to the preparation method of test solution in Example 1, and detect negative control solution and test solution according to the chromatographic conditions under Example 1.

[0197] Test results see Figure 14-15 As can be seen from the figure, there is no interference in the negative, and the method has good specificity.

[0198] 5. Stability test

[0199] According to the preparation method of the test solution in Example 1, the same batch of lyophilized powder of the standard decoction of crane lice was used to prepare the test solution and the test was performed according to the liquid phase conditions in Example 1. The test solution was injected at 0, 2, 4, 8, 12, and 24 hours after preparation, and the retention time of the common peak was recorded and the relative retention time was calculated.

[0200] The results are shown in Table 22. From the results, it can be seen that the relative retention time of each characteristic peak and the reference S peak is less than 2%, indicating that the test solution is stable within 24 hours and meets the measurement requirements.

[0201] Table 22 Stability relative retention time test results

[0202]

[0203] 6. Durability test

[0204] A. Investigation of different flow rates

[0205] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test solution was sampled and tested basically according to the chromatographic conditions in Example 1, except that the measurement was carried out at flow rates of 0.23 ml / min, 0.25 ml / min, and 0.27 ml / min to examine the separation effect of each characteristic peak of this product when the flow rate changes.

[0206] The results are shown in Table 23. From the results, it can be seen that at different flow rates, each chromatographic peak was detected and the separation effect was good.

[0207] Table 23 Relative retention time results at different flow rates

[0208]

[0209]

[0210] B. Investigation of different column temperatures

[0211] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test solution was sampled and tested basically according to the chromatographic conditions in Example 1, with the only difference that the measurement was carried out at column temperatures of 27°C, 30°C, and 33°C to examine the separation effect of each characteristic peak of this product when the column temperature changes.

[0212] The results are shown in Table 24. From the results, it can be seen that under different column temperatures, each chromatographic peak is detected and the separation effect is good.

[0213] Table 24 Comparison of relative retention time determination results at different column temperatures

[0214]

[0215] C. Investigation of different instruments

[0216] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test solution was sampled and tested basically according to the chromatographic conditions in Example 1, with the only difference that the detection was carried out on a Waters instrument and a Thermo ultra-high performance liquid chromatography, respectively, to examine the separation effect of each characteristic peak of different instruments.

[0217] The results are shown in Table 25. From the results, it can be seen that the chromatographic peaks on different instruments are all detected and the peak separation effect is good.

[0218] Table 25 Comparison of relative retention time determination results of different instruments

[0219]

[0220]

[0221] D. Investigation of different chromatographic columns

[0222] The test solution was prepared according to the preparation method of the test solution in Example 1, and the test solution was sampled and tested basically according to the chromatographic conditions in Example 1, with the only difference that Waters CORTECS T3, 100 mm × 2.1 mm, 1.6 μm and Agilent EclipsePlus C18 RRHD, 100 mm × 2.1 mm, 1.8 μm chromatographic columns were used for sample injection and analysis.

[0223] The results are shown in Table 26. From the results, it can be seen that the separation effect of each characteristic peak in the spectrum corresponding to different chromatographic columns is good.

[0224] Table 26 Comparison of relative retention time determination results of different chromatographic columns

[0225]

[0226] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of a louse and its preparation, characterized in that: The following steps are involved: (1) Preparation of test solution; (2) The sample solution is tested by ultra-high performance liquid chromatography, the filler is octadecylsilane bonded silica gel, and the mobile phase includes methanol and acid-containing aqueous solution; Gradient elution, the gradient elution program includes: 0→12 minutes→18 minutes→20 minutes→25 minutes→28 minutes→34 minutes→42 minutes, and the volume percentage of methanol in the mobile phase is 7%→18%→30%→35%→40%→45%→47%→60%.

2. The construction method according to claim 1, characterized in that: Step (1) comprises: 1) Take the object to be tested, add a solvent to extract it, and obtain an extract; 2) Separate the solid and liquid of the extract and take the liquid, which is the test solution; Preferably, step (1) also satisfies any one or more of the following AE: A. In step 1), the solvent is selected from at least one of water, methanol and ethanol; B. In step 1), the ratio of the mass of the analyte to the volume of the solvent is 0.1-2 g:10-250 mL; C. In step 1), the extraction method is reflux extraction or ultrasonic extraction, and the extraction time is 15min-120min; D. In step 2), the solid-liquid separation is centrifugation or filtration; E. In step 1), the analyte is selected from one or more of the following: Chinese lice medicinal materials, Chinese lice decoction pieces, Chinese lice granules, Chinese lice decoctions, and Chinese lice powders.

3. The construction method according to claim 1, characterized in that: The acid-containing aqueous solution is selected from at least one of a formic acid aqueous solution and a phosphoric acid aqueous solution; preferably, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%; preferably, the volume percentage of formic acid in the formic acid aqueous solution is 0.05-0.1%.

4. The construction method according to any one of claims 1 to 3, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography in step (2) also include: a detection wavelength of 210-280 nm, a flow rate of 0.23-0.27 ml / min, a column temperature of 25-33° C., a chromatographic column with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6-1.8 μm; preferably, the detection wavelength is 240 nm; preferably, the column temperature is 30° C.; preferably, the injection volume is 2 μl.

5. The construction method according to any one of claims 1 to 4, characterized in that: The gradient elution program also includes, 42 minutes→43 minutes, the volume percentage of methanol in the mobile phase is 60%→7%, or the gradient elution program also includes, 42 minutes→43 minutes→48 minutes, the volume percentage of methanol in the mobile phase is 60%→7%→7%.

6. The construction method according to any one of claims 1 to 5, characterized in that: The construction method also includes the step of preparing a reference solution using at least one reference substance selected from the group consisting of neochlorogenic acid, caffeic acid, chlorogenic acid, cryptochlorogenic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, cosmos glycoside, and asparagus lactone, and the step of obtaining a reference spectrum of the reference substance by detecting the reference solution using ultra-high performance liquid chromatography in the construction method according to any one of claims 1 to 5.

7. The construction method according to any one of claims 1 to 6, characterized in that: The characteristic spectrum of the crane louse and its preparation obtained by the construction method is selected from any one of the following (1)-(2): (1) The characteristic spectrum of the louse and its preparation has 13 characteristic peaks, with peak 9 as the reference peak, and the relative retention times of characteristic peaks 1-8 and peaks 10-13 and the reference peak are within the range of ±10% of the specified values; the specified values ​​are: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), 1.39 (peak 13); (2) The characteristic spectrum of the described sphaerocephala and its preparation has 13 characteristic peaks, the peak corresponding to the peak of the 1,5-dicaffeoylquinic acid reference substance is the reference peak, and the relative retention time of the characteristic peaks 1-8 and peaks 10-13 and the reference peak is within the range of ±10% of the specified value; the specified values ​​are: 0.21 (peak 1), 0.25 (peak 2), 0.28 (peak 3), 0.48 (peak 4), 0.49 (peak 5), 0.56 (peak 6), 0.71 (peak 7), 0.93 (peak 8), 1.06 (peak 10), 1.09 (peak 11), 1.24 (peak 12), and 1.39 (peak 13).

8. Application of the construction method described in any one of claims 1 to 7 in the quality control of lice and their preparations.

9. A method for quality control of lice and its preparation, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the crane louse product to be tested with the characteristic spectrum of the crane louse and its preparation; the characteristic spectrum of the crane louse product to be tested is obtained by using the crane louse product to be tested according to the construction method described in any one of claims 1 to 6, and the characteristic spectrum of the crane louse and its preparation is the characteristic spectrum of the crane louse and its preparation described in claim 7.

10. A method for distinguishing louse and its preparation from counterfeits, characterized in that: include: Using the product to be identified as a test sample, constructing a characteristic spectrum of the product to be identified according to the construction method described in any one of claims 1 to 6; Comparing the obtained characteristic spectrum of the product to be identified with the characteristic spectrum constructed by the construction method described in any one of claims 1 to 6, and performing identification according to the comparison result; The counterfeit products are southern crane louse and / or southern crane louse preparations.

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