Construction method and application of mistletoe formula granule characteristic chromatogram

Abstract

The invention provides a construction method and application of a specific chromatogram of mistletoe formula granules, and the construction method of the specific chromatogram of the mistletoe formula granules comprises the following steps: obtaining the specific chromatogram of a to-be-detected substance by adopting a high performance liquid chromatography; specific chromatographic conditions of the high performance liquid chromatography are provided, and octadecylsilane chemically bonded silica is used as a chromatographic column of a filler; the detection wavelength is 248 to 252nm; methanol is taken as a mobile phase A, 0.09-0.11% phosphoric acid aqueous solution is taken as a mobile phase B, and elution is carried out according to a specific gradient program. The characteristic chromatogram obtained by the construction method provided by the invention is comprehensive in material information, and fully characterizes key characteristic chemical components of the mistletoe preparation and the common adulterants of the mistletoe preparation, and the difference chemical components of the mistletoe preparation and the adulterants of the mistletoe preparation represented by the method and the adulterants of the mistletoe preparation are different. The overall quality control of the mistletoe and the identification of the mistletoe adulterants can be realized.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine identification, and in particular to a method for constructing a characteristic spectrum of mistletoe formula particles and application thereof. Background Art

[0002] Mistletoe is the dried leafy stems and branches of Viscum coloratum (Komar.) Nakai, a plant of the Loranthaceae family. It tastes bitter and flat; it enters the liver and kidney meridians.

[0003] Mistletoe has a complex origin and is a semi-parasitic plant. Although all the commercially available medicinal materials are mistletoe, they may actually be medicinal materials of different species in the same family, and there may be problems such as adulteration, which affect the effectiveness and even safety of mistletoe use to varying degrees. Commonly confused products include flat-branch mistletoe, also known as maple mistletoe (Yunnan flat-branch mistletoe, Sichuan flat-branch mistletoe), mistletoe and black mistletoe. Flat-branch mistletoe is the branches and leaves of Viscum liquidambaricolum Hayata, a plant of the Loranaceae family; mistletoe is the dried leafy stems and branches of Taxillus chinensis (DC.) Danser, a plant of the Loranaceae family; and black mistletoe is the most common confused product of mistletoe on the market. These varieties are in the same family as mistletoe and have similar names and properties, and are often adulterated.

[0004] At present, the commonly used identification methods are plant ecology identification and medicinal material appearance trait identification, which require the appraiser to have rich experience in medicinal material identification. However, after being prepared into preparations, especially formula granules, the medicinal materials lose their original appearance characteristics. Whether the preparations are derived from mistletoe or are adulterated with other counterfeits is difficult to determine with existing technology. The quality control of mistletoe preparations lacks monitoring, which affects the quality stability and effectiveness of mistletoe preparations. Summary of the invention

[0005] The characteristic spectrum construction method and application disclosed in the prior art cannot solve the problem of identifying mistletoe and its adulterants; therefore, the technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of a mistletoe preparation that can be used to identify mistletoe and its adulterants. The present invention also provides the application of the characteristic spectrum construction method in the identification of mistletoe and its adulterants, as well as the application of the characteristic spectrum construction method in the quality control of mistletoe.

[0006] A method for constructing a characteristic spectrum of a mistletoe preparation, using high performance liquid chromatography to obtain a characteristic spectrum of a test object;

[0007] The chromatographic conditions of the described high performance liquid chromatography are:

[0008] The chromatographic column was filled with octadecylsilane bonded silica gel; the detection wavelength was 248-252 nm; methanol was used as mobile phase A, 0.09-0.11% phosphoric acid aqueous solution was used as mobile phase B, and elution was performed according to the following gradient elution program:

[0009]

[0010] In the chromatographic conditions of the high performance liquid chromatography, the column length is 100 mm, the inner diameter of the column is 2.1 mm, and the particle size is 1.6 to 1.8 μm;

[0011] and / or, the chromatographic column is ACQUITY UPLC HSS T3, CORTECS UPLC T3 or Syncronis aQ;

[0012] and / or, the column temperature is 27-33°C;

[0013] and / or, a flow rate of 0.12-0.18 ml / min;

[0014] And / or, the number of theoretical plates calculated based on the syringin peak should not be less than 10,000.

[0015] When the analyte includes a mistletoe preparation, the preparation process of the analyte sample is as follows: taking the analyte, accurately weighing, adding a solvent, weighing, ultrasonic treatment, taking out, cooling, weighing again, supplementing the lost weight with a corresponding solvent, shaking, filtering, and taking a filtrate to obtain a test solution;

[0016] When the test object is a reference medicinal material, the preparation process of the test sample is as follows: taking the reference medicinal material, adding water, heat reflux treatment, cooling, filtering, and continuing to filtrate to obtain a reference medicinal material solution;

[0017] When the test substance is a reference substance, the reference substance is taken, accurately weighed, and water is added to prepare a reference substance solution containing 20 μg of the reference substance per 1 ml; the reference substances include uridine, adenosine, guanosine, thymidine, ferulic acid, caffeic acid and syringin.

[0018] The mistletoe preparation includes mistletoe water extract, mistletoe water extract dry powder, mistletoe formula granules or mistletoe water extract preparation;

[0019] And / or, the solvent is water or methanol aqueous solution.

[0020] The characteristic spectrum includes characteristic peaks from peak 1 to peak 9;

[0021] The characteristic peak corresponding to adenosine is taken as peak 2. Based on peak 2, the relative retention times of peak 1, peak 3 and peak 4 are 0.91±10%, 1.14±10% and 1.45±10%, respectively.

[0022] The characteristic peak corresponding to syringin is peak 8. Based on peak 8, the relative retention times of peaks 5 to 7 and peak 9 are 0.53±10%, 0.91±10%, 0.94±10% and 1.12±10%, respectively.

[0023] A method for constructing a characteristic spectrum of mistletoe preparations and its application in the quality control of mistletoe preparations.

[0024] An application of a characteristic spectrum of a mistletoe preparation in determining adulterated mistletoe, wherein the characteristic spectrum is obtained by using the above-mentioned construction method;

[0025] In the characteristic spectrum, the characteristic peak corresponding to the retention time of thymidine is peak a, the characteristic peak corresponding to the retention time of adenosine is peak 2, the characteristic peak corresponding to the retention time of caffeic acid is peak 7, the peak at a position 0.87±5% relative to peak 7 is peak b, the peak at a position 0.53±10% relative to peak 8 is peak 5, and the peak at a position 1.12±10% relative to peak 8 is peak 9;

[0026] The ratio of the relative peak area of ​​peak b to peak 7 is K b7 The ratio of the relative peak area of ​​peak a to peak 2 is K a2 , the ratio of the relative peak area of ​​peak 5 to peak 9 is K 59 ;

[0027] K b7 ≤0.20, K a2 ≤0.45 and K 59 ≥0.60 is mistletoe, and the rest are mistletoe adulterants.

[0028] K b7 When it is greater than 0.20, it is an adulterated product of Viscum album.

[0029] K b7 ≤0.20 and K a2 When it is greater than 0.45, it is an adulterated product of mistletoe.

[0030] K b7 ≤0.20, K a2 ≤0.45 and K 59 When <0.60, it is a black parasitic adulterated product.

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

[0032] 1. The present invention provides a method for constructing a characteristic spectrum of a mistletoe preparation. The characteristic spectrum obtained by the construction method has comprehensive material information and fully characterizes the key characteristic chemical components of the mistletoe preparation and its common adulterated products and water extract preparations. The differential chemical components of the mistletoe and its preparations and different adulterated products characterized by the method can realize the overall quality control of mistletoe and the identification of mistletoe adulterated products.

[0033] 2. In the application of the present invention, the characteristic spectrum obtained by the construction method of the present invention has comprehensive material information, covering the different chemical components of mistletoe and its preparations and different adulterated products, and therefore can be effectively applied to the overall quality control of mistletoe.

[0034] 3. In the application of the present invention, in the characteristic spectrum obtained by the construction method of the present invention, K b7 , K a2 and K 59 It can effectively realize the analysis and identification of mistletoe and its adulterants, and effectively distinguish between mistletoe, flat-branch mistletoe adulterants, mistletoe adulterants and black mistletoe adulterants. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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.

[0036] Figure 1 is the characteristic spectrum of the mistletoe in Example 1 of the present invention;

[0037] Figure 2 It is a positioning comparison diagram of peak a and peak b in the characteristic spectrum of the present invention.

[0038] Figure 3 It is a characteristic spectrum comparison diagram of the mistletoe of the present invention and its adulterated product.

[0039] Figure 4 It is the characteristic spectrum of the specificity research in Example 3 of the present invention.

[0040] Figure 5 This is the characteristic spectrum of the delay test in Example 3 of the present invention.

[0041] Figure 6 It is the characteristic spectrum of comparative example 1 of the present invention.

[0042] Figure 7 It is the characteristic spectrum of comparative example 2 of the present invention. DETAILED DESCRIPTION

[0043] 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.

[0044] Instruments: ME104E electronic balance (Mettler Toledo), JY2002 electronic balance (Mettler Toledo), DZKW-4 electronic constant temperature water bath (Beijing Zhongxing Weiye Instrument Co., Ltd.); KQ-300DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0045] Test drugs: mistletoe control medicinal materials (batch number: 121038-201906, China Food and Drug Inspection Institute), mistletoe medicinal materials (15 batches, batch numbers: HJS01-HJS15); flat-branch mistletoe medicinal materials (3 batches, batch numbers: BZHJS01-BZHJS03); mistletoe medicinal materials (3 batches, batch numbers: SJS01-SJS03); black parasitic medicinal materials (3 batches, batch numbers: black JS01-black JS03), the batch numbers and sources are shown in Table 1 below;

[0046] Table 1

[0047]

[0048] Uridine, adenosine, guanosine, thymidine, ferulic acid, caffeic acid, and syringin reference substances were purchased from the China Food and Drug Administration.

[0049] Reagents: Methanol (Merck) and phosphoric acid (Fisher Chemical) were of chromatographic grade, methanol and ethanol were of analytical grade, and water was distilled water from Watsons.

[0050] Example 1

[0051] A method for constructing a characteristic spectrum of a mistletoe preparation, the specific process is as follows:

[0052] 1. Preparation of test samples:

[0053] Pretreatment (processing) of medicinal materials: According to the method under the mistletoe decoction slices (processing) in the 2020 edition of the "Pharmacopoeia of the People's Republic of China", take mistletoe medicinal materials, mistletoe medicinal materials, flat-branch mistletoe medicinal materials and black parasite medicinal materials, remove impurities, wash them slightly, moisten them thoroughly, cut them into thick slices, and dry them to obtain mistletoe decoction slices, mistletoe decoction slices, flat-branch mistletoe decoction slices and black parasite decoction slices.

[0054] Preparation method of water-extracted dry powder (freeze-dried powder) of standard decoction: take 100g of mistletoe slices, put them in a casserole, soak them for 30 minutes, add 10 times of water for the first decoction, boil them over high heat, then simmer for 30 minutes over low heat, filter them while hot, and set aside; add 8 times of water for the second decoction, boil them over high heat, then simmer for 20 minutes over low heat, and filter them while hot; combine the filtrates, concentrate (concentration temperature ≤65°C), concentrate to a solid-liquid ratio of about 1:1, transfer the concentrate to a freeze-drying plate, put it in a freeze dryer, and freeze-dry to obtain mistletoe water-extracted dry powder (mistletoe freeze-dried powder); take the remaining mixed product slices in the same way to obtain the corresponding flat-branch mistletoe water-extracted dry powder (flat-branch mistletoe freeze-dried powder), mistletoe water-extracted dry powder (mistletoe freeze-dried powder) and black parasite water-extracted dry powder (black parasite freeze-dried powder).

[0055] The preparation method of the formula granules is as follows: 4000g of mistletoe slices are taken, extracted twice, 14 times of water and 12 times of water are added respectively, each time boiling for 0.5 hour, filtered, the filtrate is combined, and concentrated under reduced pressure to a density of 1.05-1.10, spray dried, an appropriate amount of auxiliary materials are added, mixed, and dry granulated to obtain mistletoe formula granules; the remaining mixed product slices are taken in the same way to obtain the corresponding flat-branch mistletoe formula granules, mistletoe formula granules and black parasite formula granules.

[0056] 2. Characteristic spectrum of the test product

[0057] 2.1 Solution preparation

[0058] Preparation of test solution: Take about 0.2 g of sample (mistletoe freeze-dried powder, flat-branch mistletoe freeze-dried powder, mistletoe freeze-dried powder, black parasite freeze-dried powder, mistletoe formula granules, flat-branch mistletoe formula granules, mistletoe formula granules or black parasite formula granules), accurately weigh, place in a stoppered conical flask, accurately add 15 ml of 10% methanol, weigh the weight, ultrasonically treat for 30 minutes, take out, cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0059] Preparation of reference medicinal material solution: Take 0.5 g of mistletoe reference medicinal material, place it in a stoppered conical flask, add 15 mL of water, reflux for 30 minutes, cool, filter, and use the filtrate as the reference medicinal material solution;

[0060] Reference substance solution: Take appropriate amount of uridine, adenosine, guanosine, thymidine, ferulic acid, caffeic acid and syringin, weigh accurately, add water to make a solution containing 20 μg of reference substance per 1 ml, as the reference substance solution.

[0061] 2.2 Chromatographic method

[0062] The following chromatographic conditions were used for detection:

[0063] Octadecylsilane bonded silica gel was used as filler (column length 100 mm, column inner diameter 2.1 mm, particle size 1.8 μm, ACQUITY UPLC HSS T3); methanol (chromatographically pure) was used as mobile phase A, and a 0.10% volume concentration of phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 2 below; the detection wavelength was 250 nm; the column temperature was 30° C.; the flow rate was 0.15 ml / min. The theoretical plate number should not be less than 10,000 based on the syringin peak.

[0064] Table 2

[0065]

[0066] Accurately pipette 1ul of the test solution, control medicinal material reference solution and reference substance solution respectively and inject them into the liquid chromatograph for determination.

[0067] Among them, the test solution adopts 15 batches of mistletoe medicinal materials shown in Table 1, which are processed according to the above-mentioned standard decoction water extraction dry powder (lyophilized powder) preparation method to prepare mistletoe freeze-dried powder and 3 batches of mistletoe formula granules prepared according to the above-mentioned formula granule preparation method. The test results are shown in Table 3 below.

[0068] Table 3

[0069]

[0070]

[0071] In the characteristic spectra of 15 batches of mistletoe freeze-dried powder and 3 batches of mistletoe formula granules obtained in this example, the relative retention time difference of each characteristic peak is small, and the RSD range is 0.26% to 3.19%. At the same time, the characteristic spectra of 15 batches of mistletoe freeze-dried powder and 3 batches of mistletoe formula granules are calculated for similarity, and the similarity range is 0.898 to 0.998, which is highly correlated and meets the quality control requirements.

[0072] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.1 version) was used to calculate the median and match the full spectrum peaks. Figure 1 The characteristic spectrum shown.

[0073] Figure 1In the characteristic spectrum, 9 characteristic peaks should be presented, among which the retention times of peak 1, peak 2, peak 4, peak 6, peak 7 and peak 8 should correspond to the reference peak retention times of uridine, adenosine, guanosine, ferulic acid, caffeic acid and syringin reference substances, respectively. The peak (peak 2) corresponding to the reference peak retention time of adenosine reference substance is counted as S1 peak, and the relative retention time of peak 1-peak 4 and S1 peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.91 (peak 1), 1.14 (peak 3), 1.45 (peak 4); the peak (peak 8) corresponding to the reference peak retention time of syringin reference substance is counted as S2 peak, and the relative retention time of peak 5-peak 9 and S2 peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.53 (peak 5), 0.91 (peak 6), 0.94 (peak 7), 1.12 (peak 9), and the allowable error is ±10%.

[0074] Example 2

[0075] A method for constructing a characteristic spectrum of a mistletoe preparation is used in determining mistletoe adulteration. The characteristic spectrum of the test sample is obtained by the same construction method as in Example 1, and the peak areas of peak 2, peak 5, peak 7, and peak 9 in the characteristic spectrum are obtained. The positions of peak a and peak b and the peak areas of peak a and peak b are also marked.

[0076] The positioning of peak a and peak b is as follows: the retention time of peak a is consistent with the retention time of the chromatographic peak of the thymidine reference substance, and the average relative retention time of peak b and peak 7 (caffeic acid) is 0.87, such as Figure 2 Three batches of mistletoe freeze-dried powder and three batches of mistletoe formula granules were used to verify the position of peak b, and the verification results are shown in Table 4 below.

[0077] Table 4

[0078]

[0079] As can be seen from Table 4 above, the average relative retention time between peak b and peak 7 (caffeic acid) is 0.87, and the RSD value is 0.23%. Therefore, the position of peak b is determined to be 0.87 relative to the retention time of peak 7, with an allowable error of ±5%.

[0080] Characteristic spectra of freeze-dried powder and granules of mistletoe, freeze-dried powder and granules of flat mistletoe, freeze-dried powder and granules of mistletoe, freeze-dried powder and granules of black parasite were detected, and the difference peaks of the spectra were analyzed and K was calculated. a2 , K b7 and K 59 Relative peak area ratio, the results are as follows Figure 3 As shown in Tables 5 to 8 below.

[0081] Table 5 Freeze-dried mistletoe powder and formula granules Ka2 , K b7 and K 59 result

[0082]

[0083] Results Analysis: Freeze-dried Mistletoe Powder and Formula Granules K a2 The range was 0.045 to 0.326, and the mean + 3SD value was 0.421. The K a2 ≤0.45; Mistletoe freeze-dried powder and formula granules K b7 The range was 0.0 to 0.192, and the mean + 30% value was 0.121. The K of mistletoe freeze-dried powder and formula granules was determined. b7 ≤0.20; Mistletoe freeze-dried powder and formula granules K 59 The range was 0.618 to 2.917, and the mean-3SD value was 0.701. The K 59 ≥0.60. Therefore, the above-mentioned mistletoe freeze-dried powder and formula granules in the present invention all meet K b7 ≤0.20, K a2 ≤0.45 and K 59 ≥0.60, so it can be determined that the above 15 batches of mistletoe freeze-dried powder and 3 batches of mistletoe formula granules are consistent with the actual results and are all mistletoe.

[0084] The difference peaks between Viscum album and Viscum album are peak a, peak 9, and peak b, and peak 9 is missing. The relevant peak area results are shown in Table 6:

[0085] Table 6 Viscum planatum freeze-dried powder and formula granules K a2 and K b7 result

[0086]

[0087]

[0088] Results analysis: Viscum planatum freeze-dried powder and formula granules K b7 The range was 0.364-0.704, the mean was 0.484, and the mean-1.5SD value was 0.227. The K values ​​of Viscum planulatum freeze-dried powder and formula granules were determined. b7 >0.25; Viscum album freeze-dried powder and formula granules K a2 The range is 3.38~5.40. As can be seen from the above, the K b7 Meet K b7 >0.2, which proves that it is consistent with the actual result and is an adulterated product of Viscum planulatum.

[0089] The difference peaks between black parasite and mistletoe are peak 5, peak 7, peak 8 and peak 9, and peak b is not detected. The relevant peak area results are shown in Table 7 below:

[0090] Table 7 Lyophilized powder and formula granules K of black parasite a2 and K 59 result

[0091]

[0092] Result analysis: Black parasite freeze-dried powder and formula granules K 59 The actual range is 0.209-0.320, the mean is 0.273, and the mean + 30% value is 0.355. The K value of black parasite freeze-dried powder and formula granules is determined 59 <0.35; Black parasite freeze-dried powder and formula granules K a2 The range is 0.093~0.162, and the K of black parasite freeze-dried powder and formula granules is determined a2 ≤0.2; Since peak b was not detected, K b7 It is about 0. As can be seen from the above, the K of the above-mentioned black parasite freeze-dried powder and formula granules is a2 , K 59 and K b7 Meet K b7 ≤0.20, K a2 ≤0.45 and K 59 The requirement of <0.60 proves that it is consistent with the actual results and is a black parasitic adulteration product.

[0093] The difference peak between Viscum album and Viscum album is peak a, peak 5, peak 7, peak 8, peak b and peak 9 are missing, and the relevant peak area results are as follows:

[0094] Table 8 Lyophilized powder and formula granules K a2 result

[0095]

[0096]

[0097] Result analysis: Freeze-dried powder and formula granules K a2 The actual range is 10.522~19.273, the mean is 14.88, and the mean-30% value is 10.419. a2 ≥10.0, while peak b and peak 5 are missing, it can be determined that K 59 , K b7 It is about 0. As can be seen from the above, the K a2 and K b7 Meet K b7 ≤0.20 and K a2>0.45, which proves that it is consistent with the actual results and is an adulterated product of mistletoe.

[0098] Example 3

[0099] This example is used to perform methodological verification on the chromatographic conditions in Example 1.

[0100] 1. Exclusive research

[0101] Take 0.2g of dextrin and mistletoe granules respectively, accurately add 15ml of 10% methanol, and prepare the test solution according to the method in the main text; take the test solution of the granules and the negative control solution according to the method in the main text to obtain their chromatograms, and the results are shown in the figure. Figure 4 .

[0102] Summary: The spectrum shows that the negative solution has no interference with the characteristic peaks, and this method has good specificity.

[0103] 2. Delay test

[0104] Take the mistletoe formula granule test solution, inject it according to the chromatographic conditions of the method in the text, and record the chromatogram of 2 times the retention time of the mobile phase. The results are shown in Figure 5 .

[0105] Summary: The results in the above figure show that there is no obvious lag peak after 50 minutes, and this chromatographic method has good delay performance.

[0106] Example 4

[0107] A method for constructing a characteristic spectrum of a mistletoe preparation is different from that of Example 1 in that the test solution is prepared differently.

[0108] The preparation method of the test solution is as follows: take about 0.2g of mistletoe formula granules, weigh accurately, place in a stoppered conical bottle, add water and 15ml of 10% methanol accurately, stopper, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. Inject and test according to the method determined in the text, and compare the peak areas of characteristic peaks, see Table 9.

[0109] Table 9 Extraction solvent research results

[0110]

[0111] Summary: Under the conditions of 10% methanol and water as extraction solvents, the peak areas of the characteristic peaks were not much different. Considering that the 10% methanol solution had better storage stability, the extraction solvent for the characteristic spectrum of mistletoe formula granules was determined to be 10% methanol.

[0112] Example 5

[0113] A method for constructing a characteristic spectrum of a mistletoe preparation is different from that of Example 1 in that the chromatographic conditions are different, as follows:

[0114] (1) Investigation of different flow rates

[0115] Take an appropriate amount of mistletoe freeze-dried powder, grind it into powder, take 0.2 g, prepare the test sample according to the preparation method of the test sample solution in Example 1, and measure it at flow rates of 0.12 ml / min, 0.15 ml / min and 0.18 ml / min respectively. The results are shown in Table 10.

[0116] Table 10 Relative retention time and relative peak area determination results of characteristic spectra at different flow rates

[0117]

[0118]

[0119] Summary: The data show that under the above-mentioned different flow rate conditions, the RSD range of the relative retention time of each characteristic peak in the characteristic spectrum is 0.13% to 4.24%; the RSD range of the relative peak area of ​​each characteristic peak in the characteristic spectrum is 0.34% to 6.93%, both of which are less than 10%; this method has good durability for the characteristic peak flow rate of 0.12-0.18ml / min.

[0120] (2) Investigation of different column temperatures

[0121] Take an appropriate amount of mistletoe freeze-dried powder, grind it into powder, take 0.2 g, prepare the test sample according to the preparation method of the test solution in Example 1, and measure it at column temperatures of 27°C, 30°C and 33°C respectively. The results are shown in Table 11.

[0122] Table 11 Determination results of relative retention time and relative peak area of ​​characteristic spectra at different column temperatures

[0123]

[0124] Summary: The data show that under different column temperature conditions, the relative retention time RSD of each characteristic peak in the characteristic spectrum is in the range of 0.13-1.03; the relative peak area RSD of each characteristic peak is in the range of 0.32% to 6.97%, all less than 10%. This method has good durability for column temperatures between 27-33°C.

[0125] (3) Investigation of different chromatographic columns and different detection wavelengths

[0126] Take an appropriate amount of mistletoe freeze-dried powder, grind it into powder, take 0.2 g, prepare the test sample according to the preparation method of the test sample solution in Example 1, and use chromatographic column 1: CORTECS UPLC T3 (2.1×100mm, 1.6μm), chromatographic column 2: ACQUITY UPLC HSST3 (2.1×100mm, 1.8um), chromatographic column 3: Syncronis aQ (2.1×100mm, 1.7um) to measure at detection wavelengths of 248nm, 250nm and 252nm, respectively. The results are shown in Table 12.

[0127] Table 12 Determination results of relative retention time and relative peak area of ​​characteristic spectra of different chromatographic columns and detection wavelengths

[0128]

[0129] Summary: The data showed that under different chromatographic columns and different detection wavelengths, the RSD range of the relative retention time of each characteristic peak in the characteristic spectrum was 1.11% to 5.78%; the RSD range of the relative peak area of ​​each characteristic peak in the characteristic spectrum was 3.41% to 7.98%, both of which were less than 10%; the method had good durability of characteristic peaks under the conditions of CORTECS UPLC T3 (2.1×100mm, 1.6μm), ACQUITY UPLC HSST3 (2.1×100mm, 1.8um), and Syncronis aQ (2.1×100mm, 1.7um) chromatographic columns and 248nm-252nm detection wavelength.

[0130] (4) Investigation of different phosphoric acid concentrations

[0131] Take an appropriate amount of mistletoe freeze-dried powder, grind it into powder, take 0.2 g, prepare the test sample according to the preparation method of the test sample solution in Example 1, and measure it under the conditions of 0.09%, 0.1% and 0.11% phosphoric acid concentration, respectively. The results are shown in Table 13.

[0132] Table 13 Relative retention time and relative peak area determination results of characteristic spectra of different phosphoric acid concentrations

[0133]

[0134] Summary: The data show that under different phosphoric acid concentration conditions, the relative retention time RSD of each characteristic peak in the characteristic spectrum is in the range of 0.18-1.51; the relative peak area RSD of each characteristic peak ranges from 1.21% to 5.50%, all less than 10%. This method has good durability for phosphoric acid concentrations between 0.09% and 0.11%.

[0135] Comparative Example 1

[0136] A method for constructing a characteristic spectrum of a mistletoe preparation, which differs from Example 1 in that the high performance liquid chromatography method is different, is as follows:

[0137] Waters ACQUITY UPLC○RBEH Shield RP18 chromatographic column (2.1*100mm, 1.7um) was used, with acetonitrile-0.1% phosphoric acid solution as the mobile phase; gradient elution was performed according to the provisions in Table 14, the detection wavelength was 210nm, the column temperature was 30°C, and the flow rate was 0.3ml / min.

[0138] Table 14

[0139]

[0140] The spectrum obtained under this condition is as follows Figure 6 As shown, except for one larger peak at 210 nm, the other chromatographic peaks are smaller, and the peak shape and separation are not good; and the chromatographic peak of syringin is smaller, poorly separated and has a poor peak shape.

[0141] Comparative Example 2

[0142] A method for constructing a characteristic spectrum of a mistletoe preparation, which differs from Example 1 in that the high performance liquid chromatography method is different, is as follows:

[0143] Waters ACQUITY HSS T3 (2.1*100mm, 1.8μm) chromatographic column, acetonitrile-0.1% formic acid solution as mobile phase; gradient elution as specified in Table 15. Detection wavelength is 250nm, column temperature is 30℃, flow rate is 0.3ml / min.

[0144] Table 15

[0145]

[0146] The spectrum obtained under this condition is as follows Figure 7 As shown, the syringin peak is small and basically separated, but it is unstable and has poor repeatability.

[0147] 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 mistletoe preparation, characterized in that: High performance liquid chromatography is used to obtain characteristic spectra of the analyte; The chromatographic conditions of the described high performance liquid chromatography are: The chromatographic column was filled with octadecylsilane bonded silica gel; the detection wavelength was 248-252 nm; methanol was used as mobile phase A, 0.09-0.11% phosphoric acid aqueous solution was used as mobile phase B, and elution was performed according to the following gradient program:

2. The construction method according to claim 1, characterized in that: In the chromatographic conditions of the high performance liquid chromatography, the column length is 100 mm, the inner diameter of the column is 2.1 mm, and the particle size is 1.6 to 1.8 μm; and / or, the chromatographic column is ACQUITY UPLC HSS T3, CORTECS UPLC T3 or Syncronis aQ; and / or, the column temperature is 27-33°C; and / or, a flow rate of 0.12-0.18 ml / min; And / or, the number of theoretical plates calculated based on the syringin peak should not be less than 10,000.

3. The construction method according to claim 2, characterized in that When the analyte includes a mistletoe preparation, the preparation process of the analyte sample is as follows: taking the analyte, accurately weighing, adding a solvent, weighing, ultrasonic treatment, taking out, cooling, weighing again, supplementing the lost weight with a corresponding solvent, shaking, filtering, and taking a filtrate to obtain a test solution; When the test object is a reference medicinal material, the preparation process of the test sample is as follows: taking the reference medicinal material, adding water, heat reflux treatment, cooling, filtering, and continuing to filtrate to obtain a reference medicinal material solution; When the test substance is a reference substance, the reference substance is taken, accurately weighed, and water is added to prepare a reference substance solution containing 20 μg of the reference substance per 1 ml. The reference substances include uridine, adenosine, guanosine, thymidine, ferulic acid, caffeic acid and syringin.

4. The construction method according to claim 3, characterized in that: The mistletoe preparation includes mistletoe water extract, mistletoe water extract dry powder, mistletoe formula granules or mistletoe water extract preparation; And / or, the solvent is water or methanol aqueous solution.

5. The construction method according to any one of claims 1 to 4, characterized in that: The characteristic spectrum includes characteristic peaks from peak 1 to peak 9; The characteristic peak corresponding to adenosine is taken as peak 2. Based on peak 2, the relative retention times of peak 1, peak 3 and peak 4 are 0.91±10%, 1.14±10% and 1.45±10%, respectively. The characteristic peak corresponding to syringin is peak 8. Based on peak 8, the relative retention times of peaks 5 to 7 and peak 9 are 0.53±10%, 0.91±10%, 0.94±10% and 1.12±10%, respectively.

6. Application of the characteristic spectrum constructed by the construction method according to any one of claims 1 to 5 in the quality control of mistletoe preparations.

7. Application of a characteristic spectrum of a mistletoe preparation in determining adulterated mistletoe, characterized in that: Acquire a characteristic spectrum using the construction method described in any one of claims 1 to 5; In the characteristic spectrum, the characteristic peak corresponding to the retention time of thymidine is peak a, the characteristic peak corresponding to the retention time of adenosine is peak 2, the characteristic peak corresponding to the retention time of caffeic acid is peak 7, the peak at a position 0.87±5% relative to peak 7 is peak b, the peak at a position 0.53±10% relative to peak 8 is peak 5, and the peak at a position 1.12±10% relative to peak 8 is peak 9; The ratio of the relative peak area of ​​peak b to peak 7 is K b7 The ratio of the relative peak area of ​​peak a to peak 2 is K a2 , the ratio of the relative peak area of ​​peak 5 to peak 9 is K 59 ; K b7 ≤0.20, K a2 ≤0.45 and K 59 ≥0.60 is mistletoe, and the rest are mistletoe adulterants.

8. The use according to claim 7, characterized in that: K b7 When it is greater than 0.20, it is an adulterated product of Viscum album.

9. The use according to claim 7 or 8, characterized in that: K b7 ≤0.20 and K a2 When it is greater than 0.45, it is an adulterated product of mistletoe.

10. The use according to claim 7 or 8, characterized in that: K b7 ≤0.20, K a2 ≤0.45 and K 59 When <0.60, it is a black parasitic adulterated product.

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