A construction method of HPLC characteristic chromatogram of aconiti lateralis radicis preparata, aconiti lateralis radicis, standard decoction and formula granules

Abstract

The application provides a kind of hedgehog skin medicinal material, decoction piece, standard decoction and its formula granule HPLC characteristic chromatogram construction method, comprising the following steps: A) the raw material of hedgehog skin is extracted using solvent, to obtain the test solution;B) the test solution is determined using high performance liquid chromatography, to obtain the HPLC characteristic chromatogram of the raw material of hedgehog skin;The high performance liquid chromatography chromatographic conditions are as follows: the chromatographic column is C18 column;Mobile phase A is acetonitrile, mobile phase B is 0.05% acetic acid, gradient elution.The application establishes the HPLC characteristic chromatogram method of hedgehog skin (hedgehog) medicinal material, decoction piece, standard decoction and formula granule, to provide more scientific identification basis for identifying hedgehog skin medicinal material, decoction piece, standard decoction and formula granule.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of analytical detection, and particularly relates to a construction method of HPLC characteristic spectrum of Erinaceus europaeus Linnaeus. BACKGROUND

[0002] Erinaceus europaeus Linnaeus is a kind of dry skin of Erinaceus europaeus Linnaeus or Hemichianus dauricus Sundevall. Compared with Hemichianus dauricus Sundevall, Erinaceus europaeus Linnaeus is more widely distributed, has more quantity, and is easier to obtain, so the sample collected in the present research is based on Erinaceus europaeus Linnaeus. It can be captured all year round, and it is easier in winter. After being captured, the skin is peeled off, stretched with bamboo leaves or nailed on a wooden board, muscle is removed, and a layer of lime is spread on the inner side, and then it is dried in a ventilated place. It has the effects of regulating qi and relieving pain, cooling blood and stopping bleeding, reducing adverse and stopping vomiting, removing nebula and clearing vision, and retaining essence and stopping leakage. The standard decoction is a kind of freeze-dried powder prepared from medicinal materials by processing according to a fixed preparation process. In order to ensure the uniformity and stability of the quality of Erinaceus europaeus Linnaeus medicinal materials, pieces and standard decoction, a new characteristic spectrum method is established to control the quality. SUMMARY

[0003] The present application aims to provide a construction method of HPLC characteristic spectrum of Erinaceus europaeus Linnaeus medicinal materials, pieces, standard decoction and formula granules, and to provide more scientific technical means for identifying Erinaceus europaeus Linnaeus medicinal materials, pieces, standard decoction and formula granules.

[0004] The present application provides a construction method of HPLC characteristic spectrum of Erinaceus europaeus Linnaeus medicinal materials, pieces, standard decoction and formula granules, which comprises the following steps:

[0005] A) Erinaceus europaeus Linnaeus raw materials are extracted by solvent to obtain a test solution;

[0006] B) the test solution is determined by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the Erinaceus europaeus Linnaeus raw materials;

[0007] The high performance liquid chromatography has the following chromatographic conditions: a C18 column is used as the chromatographic column; acetonitrile is used as the mobile phase A, and 0.05% acetic acid is used as the mobile phase B, and gradient elution is adopted.

[0008] Preferably, the Erinaceus europaeus Linnaeus raw materials are medicinal materials, pieces, standard decoction and formula granules.

[0009] Preferably, the preparation method of the test solution is as follows:

[0010] Erinaceus europaeus Linnaeus raw materials are added into 10% methanol for ultrasonic treatment, cooled, shaken, filtered, and the filtrate is obtained.

[0011] The power of the ultrasonic treatment is 600 W, and the frequency is 40 kHz; the extraction time is 20-40 min, more preferably 30 min.

[0012] Preferably, the mass to volume ratio of the hedgehog skin standard decoction and the hedgehog skin formula granules to solvent (10% methanol) is 0.5 g:25 mL.

[0013] The mass to volume ratio of the hedgehog skin medicinal material, decoction piece to solvent is 1 g:25 mL.

[0014] In ultrasonic extraction and reflux extraction, the chromatogram effect is basically consistent, and is better than that of ultrasonic extraction after water decocting. Therefore, the present application selects ultrasonic extraction as the test sample extraction method.

[0015] Under different extraction time conditions, the chromatogram effect is basically consistent. In order to ensure sufficient extraction, the test sample extraction time is determined to be 30 minutes.

[0016] When the solvent addition amount is 25 ml, the characteristic map chromatographic peak area is moderate. Therefore, the test sample solvent addition amount is determined to be 25 ml.

[0017] When 10% methanol is used as the extraction solvent, the chromatographic peak information amount is larger, and the peak type is better.

[0018] Preferably, it also includes preparing a reference solution of the control and a reference solution of the control medicinal material;

[0019] Take uracil, guanine, hypoxanthine, xanthine, inosine and guanosine, add 10% methanol to dissolve, to obtain the reference solution of the control;

[0020] Take the hedgehog skin control medicinal material, add 10% methanol to ultrasonic extract, to obtain the reference solution of the control medicinal material.

[0021] The reference solution of the control and the reference solution of the control medicinal material are determined by high performance liquid chromatography, to obtain the chromatogram of the reference of the control and the reference of the control medicinal material respectively; and the components of the HPLC characteristic map of the hedgehog skin medicinal material, decoction piece, standard decoction and formula granules are qualitatively determined according to the chromatogram of the reference of the control and the reference of the control medicinal material.

[0022] Preferably, the chromatographic column is a C18 column with a specification of 5 μm, 4.6*250 mm; the column temperature is 20 DEG C. Under this column temperature, the resolution is good, the chromatogram peak shape is relatively symmetrical and the retention time is appropriate.

[0023] Preferably, the flow phase flow rate is 0.5 mL / min; the detection wavelength is 250 nm; the injection volume is 10 μL. The flow rate is 0.5 ml / min, the separation degree is good, the chromatogram peak shape is relatively symmetrical and the retention time is suitable; the information amount of the chromatographic peak is larger when the detection wavelength is 250 nm, and the chromatogram baseline is more stable;

[0024] Preferably, the gradient elution is specifically as follows:

[0025] 0-20 min, A phase: 0%, B phase: 100%;

[0026] 20-21 min, A phase: 0%-1%, B phase: 100%-99%;

[0027] 21-35 min, A phase: 1%, B phase: 99%;

[0028] 35-38 min, A phase: 1%-2.5%, B phase: 99%-97.5%;

[0029] 38-70 min, A phase: 2.5%, B phase: 97.5%.

[0030] Preferably, the similarity of the HPLC characteristic chromatogram of the hedgehog skin medicinal material, decoction piece, standard decoction and formula granules is evaluated by using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the HPLC standard characteristic chromatogram of the hedgehog skin medicinal material, decoction piece, standard decoction and formula granules is obtained, which is composed of 8 characteristic peaks, wherein peak 2 is a uracil peak; peak 3 (S) is a guanine peak; peak 5 is a hypoxanthine peak; peak 6 is a xanthine peak; peak 7 is an inosine peak; and peak 8 is a guanosine peak.

[0031] The peak 3, the peak 7 and the peak 8 correspond to the retention time of the corresponding reference peak of the control substance, respectively.

[0032] Preferably, in the HPLC characteristic chromatogram of the hedgehog skin medicinal material, decoction piece, standard decoction and formula granules, the guanine is taken as a reference peak S peak, and the relative retention times of the peak 1, the peak 2, the peak 4, the peak 5, the peak 6 and the S peak are calculated, which should be within ±10% of the specified value, and the specified value is 0.54 (peak 1), 0.75 (peak 2), 1.32 (peak 4), 1.46 (peak 5) and 1.67 (peak 6).

[0033] The application provides a kind of hedgehog skin medicinal materials, decoction piece, standard decoction and its formula granule HPLC characteristic spectrum construction method, comprising the following steps: A) the raw material of hedgehog skin is extracted using solvent, to obtain the test solution;B) the test solution is determined using high performance liquid chromatography, to obtain the HPLC characteristic spectrum of hedgehog skin raw material;The high performance liquid chromatography chromatographic conditions are: C18 column is used as chromatographic column;Mobile phase A is acetonitrile, mobile phase B is 0.05% acetic acid, gradient elution.The application establishes the HPLC characteristic spectrum method of hedgehog skin (hedgehog) medicinal materials, decoction piece, standard decoction and formula granule, to provide more scientific identification basis for identifying hedgehog skin medicinal materials, decoction piece, standard decoction and formula granule. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creating any inventive labor.

[0035] Figure 1 The standard decoction chromatogram of different wavelengths;

[0036] Figure 2 The column temperature investigation of standard decoction;

[0037] Figure 3 The flow rate investigation of standard decoction;

[0038] Figure 4 The characteristic spectrum chromatographic peak identification of scalded hedgehog skin (hedgehog) standard decoction;

[0039] Figure 5 The spectral overlay of uracil reference substance and target peak in sample;

[0040] Figure 6 The spectral overlay of guanine reference substance and target peak in sample;

[0041] Figure 7 The spectral overlay of hypoxanthine reference substance and target peak in sample;

[0042] Figure 8 The spectral overlay of xanthine reference substance and target peak in sample;

[0043] Figure 9 The spectral overlay of inosine reference substance and target peak in sample;

[0044] Figure 10 The spectral overlay of guanosine reference substance and target peak in sample;

[0045] Figure 11Investigation of different instruments for standard decoction of boiled hedgehog skin (Erinaceus europaeus Linn.);

[0046] Figure 12 Investigation of different chromatographic columns for standard decoction of boiled hedgehog skin (Erinaceus europaeus Linn.);

[0047] Figure 13 Characteristic chromatogram of standard decoction of boiled hedgehog skin (Erinaceus europaeus Linn.);

[0048] Figure 14 Control characteristic chromatogram of standard decoction;

[0049] Figure 15 Investigation of extraction solvent;

[0050] Figure 16 Investigation of extraction method;

[0051] Figure 17 Investigation of extraction time;

[0052] Figure 18 Investigation of solvent addition amount;

[0053] Figure 19 Identification of chromatographic peaks of characteristic chromatogram of Erinaceus europaeus Linn. medicinal material;

[0054] Figure 20 Investigation of different instruments for Erinaceus europaeus Linn. medicinal material;

[0055] Figure 21 Investigation of chromatographic column durability;

[0056] Figure 22 Verification chromatogram of characteristic chromatogram of 16 batches of Erinaceus europaeus Linn. medicinal material;

[0057] Figure 23 Control characteristic chromatogram of Erinaceus europaeus Linn. medicinal material;

[0058] Figure 24 Characteristic chromatogram of Erinaceus europaeus Linn. decoction piece;

[0059] Figure 25 Control characteristic chromatogram of Erinaceus europaeus Linn. decoction piece;

[0060] Figure 26 Ultraviolet absorption spectrum of uracil;

[0061] Figure 27 Ultraviolet absorption spectrum of guanine;

[0062] Figure 28 Ultraviolet absorption spectrum of hypoxanthine;

[0063] Figure 29 Ultraviolet absorption spectrum of xanthine;

[0064] Figure 30UV absorption spectrum of inosine;

[0065] Figure 31 UV absorption spectrum of guanosine;

[0066] Figure 32 Different wavelength chromatogram of scalded hedgehog skin (hedgehog) formula granules;

[0067] Figure 33 Column temperature investigation chromatogram;

[0068] Figure 34 Flow rate investigation chromatogram;

[0069] Figure 35 Extraction solvent investigation;

[0070] Figure 36 Extraction method investigation;

[0071] Figure 37 Extraction time investigation;

[0072] Figure 38 Solvent addition amount investigation;

[0073] Figure 39 Scalded hedgehog skin (hedgehog) finished product characteristic chromatogram peak identification;

[0074] Figure 40 Scalded hedgehog skin (hedgehog) finished product different instrument characteristic chromatogram comparison;

[0075] Figure 41 Chromatographic column durability investigation;

[0076] Figure 42 3 batches of scalded hedgehog skin (hedgehog) formula granules characteristic chromatogram verification diagram;

[0077] Figure 43 Scalded hedgehog skin (hedgehog) formula granules control characteristic chromatogram. DETAILED DESCRIPTION

[0078] The present application provides a kind of hedgehog skin (hedgehog) medicinal materials, decoction piece, standard decoction and formula granule HPLC characteristic chromatogram method, those skilled in the art can refer to the content of this paper, and appropriately improve process parameters to realize.It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they all belong to the scope protected by the present application.The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this paper without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0079] In order to further illustrate the present application, the following embodiments are described in detail in combination with the HPLC characteristic chromatogram method of a hedgehog skin (Erinaceus) medicinal material, decoction piece, standard decoction and formula granules provided by the present application.

[0080] Experimental instruments and materials

[0081] Instruments: 2 different brands of high performance liquid chromatographs;

[0082] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0083] Ultrapure water machine: CelluSafe 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0084] Electric heating constant temperature water bath: KQ-600DB type (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.);

[0085] Chromatographic column: 3 different types of chromatographic columns.

[0086] Acetic acid, acetonitrile are chromatographically pure, water is ultrapure, and the rest of the reagents are analytically pure.

[0087] Uracil (China Food and Drug Inspection Research Institute, batch number: 100469-201302, content is 99.6%);

[0088] Guanine (China Food and Drug Inspection Research Institute, batch number: 140631-202008, content is 98.9%);

[0089] Hypoxanthine (China Food and Drug Inspection Research Institute, batch number: 140661-202005, content is 99.4%);

[0090] Xanthine (China Institute for Drug Control, batch number: 140662-200802, no content requirement),

[0091] Inosine (China Food and Drug Inspection Research Institute, batch number: 140669-202007, content is 99.2%);

[0092] Guanylate (China Food and Drug Inspection Research Institute, batch number: 111977-202202, content is 88.6%);

[0093] Hedgehog skin (Erinaceus) control medicinal material (Shandong BoKang Fine Chemical Co., Ltd., batch number: 230156-202305),

[0094] Scorzonera hispidus (Hedgehog) standard decoction freeze-dried powder (prepared by Sichuan Xinglv Pharmaceutical Science and Technology Development Co., Ltd., batch number: TCWP-BT-230601, TCWP-BT-230601, TCWP-BT-230602, TCWP-BT-230603, TCWP-BT-230604, TCWP-BT-230605, TCWP-BT-230606, TCWP-BT-230607, TCWP-BT-230608, TCWP-BT-230609, TCWP-BT-230610, TCWP-BT-230611, TCWP-BT-230612, TCWP-BT-230613, TCWP-BT-230614, TCWP-BT-230615, TCWP-BT-230616).

[0095] Scorzonera hispidus (Hedgehog) medicinal material (prepared by Sichuan Xinglv Pharmaceutical Science and Technology Development Co., Ltd., batch number: XXLS202305729, XXLS202305730, XXLS202305731, XXLS202305732, XXLS202305733, XXLS202305734, XXLS202305735, XXLS202305736, XXLS202305737, XXLS202305738, XXLS202305739, XXLS202305740, XXLS202305741, XXLS202305742, XXLS202305743, 010091-2306001.

[0096] Scorzonera hispidus (Hedgehog) decoction pieces (prepared by Sichuan Xinglv Pharmaceutical Science and Technology Development Co., Ltd., batch number: TCWP-230601, TCWP-230601, TCWP-230602, TCWP-230603, TCWP-230604, TCWP-230605, TCWP-230606, TCWP-230607, TCWP-230608, TCWP-230609, TCWP-230610, TCWP-230611, TCWP-230612, TCWP-230613, TCWP-230614, TCWP-230615, TCWP-230616)

[0097] Scorzonera hispidus (Hedgehog) formula granules (prepared by Sichuan Xinglv Pharmaceutical Science and Technology Development Co., Ltd., batch number: 1903019, 2306093, 2306094, 2306095).

[0098] Example 1 Scorzonera hispidus (Hedgehog) standard decoction HPLC characteristic spectrum

[0099] 1.1 Chromatographic conditions and system suitability test Use octadecylsilane-bonded silica gel as the packing agent; use acetonitrile as the mobile phase A and 0.05% acetic acid solution as the mobile phase B, perform gradient elution according to the provisions in the following table; the flow rate is 0.6 ml per minute; the column temperature is 20°C; and the detection wavelength is 250 nm. The theoretical plate number should not be less than 5000 calculated according to the guanine peak.

[0100]

[0101] Preparation of reference solution Take 1 g of hedgehog skin (hedgehog) control medicinal material, place it in a conical flask with a plug, add 10% methanol 25 ml, tightly plug, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the filtrate, and use it as the control medicinal material reference solution. Take the appropriate amount of uracil, guanine, guanosine, xanthine, inosine, and guanosine reference substances, accurately weigh, add 10% methanol to prepare a solution containing 40 μg of each per 1 ml, and obtain it.

[0102] Preparation of test solution Take about 0.5 g of the product, place it in a conical flask with a plug, add 10% methanol solution 25 ml, tightly plug, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate, and obtain it.

[0103] Determination method: accurately take 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and obtain it.

[0104] 1.2 Chromatographic conditions and system suitability test

[0105] 1.2.1 Wavelength selection

[0106] On the basis of the above proposed experimental conditions, full-wave band scanning was performed on the uracil, guanine, hypoxanthine, xanthine, inosine, and guanosine solutions using a diode array detector, and the chromatograms of the test solution at 210 nm, 230 nm, 250 nm, 270 nm, 290 nm, and 310 nm wavelengths were extracted, respectively, as shown in Figure 1 .

[0107] The results show that when the detection wavelength is 250 nm, the amount of chromatographic peak information is larger, and the baseline of the chromatogram is more stable, so the detection wavelength is determined to be 250 nm.

[0108] 1.2.2 Column temperature investigation

[0109] On the basis of the above proposed experimental conditions, investigations were carried out at column temperatures of 20°C, 25°C, 30°C, and 35°C, respectively. See Figure 2 .

[0110] The results of column temperature investigation show that at different column temperatures, the column temperature of 20 °C has good separation, the peak shape of chromatogram is relatively symmetrical and the retention time is appropriate, therefore, the column temperature is selected as 20 °C.

[0111] 1.2.3 Flow rate investigation

[0112] On the basis of the above proposed experimental conditions, the flow rates of 0.5 ml / min, 0.6 ml / min and 0.7 ml / min are investigated respectively. See Figure 3 .

[0113] The results of flow rate investigation show that at different flow rates, the flow rate of 0.5 ml / min has good separation, the peak shape of chromatogram is relatively symmetrical and the retention time is appropriate, therefore, the flow rate is selected as 0.5 ml / min.

[0114] In summary, the chromatographic conditions and system suitability test of the characteristic chromatogram of the scalded hedgehog skin (hedgehog) formula granules are determined as follows: octadecylsilane-bonded silica gel is used as the filler; acetonitrile is used as the mobile phase A, and 0.05% acetic acid solution is used as the mobile phase B, which is gradient eluted according to the following table; the flow rate is 0.5 ml / min; the column temperature is 20 °C; and the detection wavelength is 250 nm. The theoretical plate number calculated according to the guanine peak should not be less than 5000.

[0115]

[0116] 1.3 Methodology investigation

[0117] 1.3.1 Identification of chromatographic peaks

[0118] Preparation of test solution: according to the above proposed experimental conditions, the test solution of the scalded hedgehog skin (hedgehog) standard decoction is prepared.

[0119] Preparation of reference solution: take a certain amount of uracil, guanine, guanosine, xanthine, inosine and guanosine reference substances, accurately weigh, add 10% methanol to prepare a solution containing 40 μg of each per 1 ml, and obtain it.

[0120] Preparation of hedgehog skin (hedgehog) control medicinal material solution: take 1 g of hedgehog skin (hedgehog) control medicinal material, put it in a conical flask with a plug, add 25 ml of 10% methanol, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake evenly, filter, take the filtrate, as the reference solution of the control medicinal material.

[0121] Preparation of negative control solution: according to the above proposed experimental conditions, the negative control solution of the scalded hedgehog skin (hedgehog) standard decoction is prepared.

[0122] The peaks of the characteristic chromatogram of the scalded hedgehog skin (hedgehog) standard decoction are located. See Figures 4-10 .

[0123] The results show that the retention time and spectrum of the control samples of uracil, guanine, hypoxanthine, xanthine, inosine and guanosine can be consistent with the retention time and spectrum of the target peaks in the Erxiongshi (Echidna) standard decoction Figure 1 One corresponds to the negative solution without interference, and the method is specific.

[0124] 1.3.2 Precision test

[0125] Take the Erxiongshi (Echidna) standard decoction test solution, continuously sample 6 times, 10 μl each time, according to the proposed experimental method, and calculate the retention time of each characteristic peak. See Table 1.

[0126] Table 1 Precision test - retention time

[0127]

[0128]

[0129] The results show that the precision of each characteristic peak retention time RSD is 0.04%-0.23%. The instrument precision is good.

[0130] 1.3.3 Reproducibility test

[0131] Precisely weigh 6 portions of Erxiongshi (Echidna) standard decoction (batch number: TCWP-BT-230501), prepare and determine according to the proposed experimental method. See Table 2.

[0132] Table 2 Reproducibility test - relative retention time ratio of characteristic peaks

[0133]

[0134] The results show that the reproducibility of each characteristic peak relative retention time is consistent, and the relative retention time RSD is 0.00%-0.81%. The method is reproducible.

[0135] 1.3.4 Intermediate precision test

[0136] Different instrument test

[0137] On the basis of the above proposed experimental conditions, 12 portions of Erxiongshi (Echidna) standard decoction were precisely weighed to prepare the test solution, which was determined on high performance liquid chromatograph 1 and 2 respectively. See Table 3. Figure 11

[0138] Table 3 Different instrument test - relative retention time ratio of characteristic peaks

[0139]

[0140] ​

[0141] The results show that the intermediate precision of the method is good.

[0142] 1.3.5 Durability of the chromatographic column

[0143] Based on the above experimental conditions, different brands of chromatographic column 1, 2, 3 were investigated. The results are shown in Table 4. Figure 12

[0144] Table 4 Durability of the chromatographic column - relative retention time ratio of characteristic peaks

[0145]

[0146] The results show that the relative retention time RSD values differ greatly when different chromatographic columns are used. It is recommended to use chromatographic column 1.

[0147] 1.3.6 Stability investigation

[0148] Based on the above experimental conditions, the same test solution was taken and measured at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h. See Table 5.

[0149] Table 5 Stability investigation - retention time

[0150]

[0151] The results show that the RSD of the corresponding characteristic peak retention time is 0.14% to 1.00%, and the sample solution is stable within 24 hours.

[0152] In summary, the RSD of the retention time / relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good.

[0153] 1.4 Determination of characteristic peaks and establishment of control chromatogram

[0154] 1.4.1 Verification results of 16 batches of boiled hedgehog skin (hedgehog) standard decoction

[0155] Using this method, the relative retention time was calculated by analyzing the characteristic chromatograms of 16 batches of samples. See Table 6. Figure 13 Figure 13 ​​TCWP-BT-230601, TCWP-BT-230602, TCWP-BT-230603, TCWP-BT-230604, TCWP-BT-230605, TCWP-BT-230606, TCWP-BT-230607, TCWP-BT-230608, TCWP-BT-230609, TCWP-BT-230610, TCWP-BT-230611, WFG-BT-210912, TCWP-BT-230613, TCWP-BT-230614, TCWP-BT-230615, TCWP-BT-230616).

[0156] Table 6 Relative retention time ratio of scalded and pickled hedgehog skin

[0157]

[0158]

[0159] According to the principle of stable relative retention time and detectable peaks, 8 characteristic peaks with good repeatability were selected. The results showed that when peak 3 was used as the S peak, the RSD of the relative retention time of peaks 1-6 was 0.00%.

[0160] 1.4.2 Establishment of the limit of the relative retention time

[0161] The results of the methodological investigation and validation are shown in Table 7:

[0162] Table 7 RSD (%) of the results of each item of the methodology

[0163]

[0164] The results showed that, except for the column durability, the RSD values of the retention time or relative retention time of peaks 1-6 were small, basically meeting the requirements; the relative retention time of peaks 7 and 8 had large differences in the intermediate precision investigation, and the subsequent reference substance was considered for identification.

[0165] The final provisions: test sample chromatography should present 8 characteristic peaks, and should be with the control drug reference chromatography of 8 characteristic peaks retention time corresponding, wherein peak 3, peak 7, peak 8 should be respectively corresponding to the retention time of the corresponding reference peak, and the peak corresponding to the guanine reference is S peak, the relative retention time of peak 1, peak 2, peak 4, peak 5, peak 6 and S peak should be within the range of ± 10% of the specified value, the specified value is: 0.54 (peak 1), 0.75 (peak 2), 1.32 (peak 4), 1.46 (peak 5), 1.67 (peak 6).

[0166] The similarity evaluation system of traditional Chinese medicine chromatographic fingerprint (2012 edition) was used to synthesize 16 batches of standard Erzhu skin decoction, and the control chromatogram of Erzhu skin standard decoction characteristic chromatogram was established. Figure 14 .

[0167] 1.5 Erzhu skin standard decoction characteristic chromatogram method

[0168] Chromatographic conditions and system suitability test with octadecylsilane bonded silica gel as filler (column length of 250 mm, inner diameter of 4.6 mm, particle size of 5 μm); with acetonitrile as mobile phase A, and 0.05% acetic acid solution as mobile phase B, gradient elution according to the following table; flow rate of 0.5 ml per minute; column temperature of 20 ℃; detection wavelength of 250 nm. The theoretical plate number should not be less than 5000 according to the guanine peak.

[0169]

[0170]

[0171] Preparation of reference solution: take Erzhu skin (Erzhu) control drug 1 g, put it in a conical flask with plug, add 10% methanol 25 ml, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake evenly, filter, take the filtrate, as the control drug reference solution. In addition, take appropriate amount of uracil, guanine, guanosine, xanthine, inosine, guanosine reference substance, accurately weigh and add 10% methanol to prepare a solution containing 40 μg per 1 ml, and then get it.

[0172] Preparation of test solution: take about 0.5 g of the product, put it in a conical flask with plug, add 10% methanol solution 25 ml, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake evenly, filter, take the filtrate, and then get it.

[0173] Determination: precisely take 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and then determine.

[0174] Example 2 HPLC characteristic chromatogram of Erzhu skin medicinal material and decoction piece

[0175] 2.1 Chromatographic conditions and system suitability Octadecylsilane-bonded silica gel as the packing agent; acetonitrile as the mobile phase A, 0.05% acetic acid solution as the mobile phase B, gradient elution according to the following table; flow rate 0.5 ml per minute; column temperature 20 °C; detection wavelength 250 nm.

[0176] Theoretical plate number should not be less than 5000 calculated according to the guanine peak.

[0177]

[0178] Preparation of reference solution About 1.0 g of hedgehog skin (hedgehog) control medicinal material was placed in a conical flask with a plug, 10% methanol 25 ml was added, and ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. After cooling, it was shaken uniformly, filtered, and the filtrate was taken as the control medicinal material reference solution. In addition, a certain amount of uracil, guanine, hypoxanthine, xanthine, inosine, and guanosine reference substances were precisely weighed and dissolved in 10% methanol to prepare a solution containing 40 μg per 1 ml, and the solution was obtained.

[0179] Preparation of test solution A certain amount of the product powder (passed through a No. 3 sieve) was taken, about 1.0 g was placed in a conical flask with a plug, 10% methanol solution 25 ml was added, the flask was tightly plugged, ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes, and after cooling, it was shaken uniformly, filtered, and the filtrate was taken as the test solution.

[0180] Determination method 10 μl of the reference solution and the test solution were precisely taken and injected into the liquid chromatograph, and determination was performed, and the test solution was obtained.

[0181] 2.2 Investigation of preparation of test solution

[0182] 2.2.1 Investigation of extraction solvent for preparation of test solution

[0183] A certain amount of the product powder (passed through a No. 3 sieve) (batch number: 010091-2306001) was taken, about 1.0 g was placed in a conical flask with a plug, water, 10% methanol, 30% methanol, 70% methanol, methanol, and 10% ethanol 25 ml were added respectively, the flask was tightly plugged, ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes, and after cooling, it was shaken uniformly, filtered, and the filtrate was taken. Figure 15 .

[0184] The results showed that when 10% methanol was used as the extraction solvent, the chromatographic peak information was larger and the peak shape was better, so the extraction solvent for the test solution was determined to be 10% methanol.

[0185] 2.2.2 Investigation of extraction method for preparation of test solution

[0186] Take the product powder (over three sieve) (batch number: 010091-2306001) appropriate amount, take about 1.0g, put in the conical flask with plug, add 10% methanol 25ml, respectively, for test sample extraction method is reflux, ultrasonic (power 600W, frequency 40kHz) for investigation, extraction time 30 minutes, cool, shake, filter, take the filter solution, namely. When water is extracted, add 25ml water, boil for half an hour, filter, evaporate to dryness, add 10% methanol 25ml to the residue, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool, shake, filter, take the filter solution, namely. See Figure 16 .

[0187] The results show that in ultrasonic extraction and reflux extraction, the chromatogram effect is basically the same, and is better than that of water decoction and ultrasonic extraction. Therefore, the ultrasonic extraction is selected as the test sample extraction method in this experiment.

[0188] 2.2.3 Test sample preparation extraction time investigation

[0189] Take the product powder (over three sieve) (batch number: 010091-2306001) appropriate amount, take about 1.0g, put in the conical flask with plug, add 10% methanol 25ml, tightly plug, respectively, for test sample ultrasonic treatment (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, 40 minutes, cool, shake, filter, take the filter solution, namely. See Figure 17 .

[0190] The results show that under different extraction time conditions, the chromatogram effect is basically the same. In order to ensure sufficient extraction, the test sample extraction time is determined to be 30 minutes.

[0191] 2.2.4 Solvent addition amount investigation

[0192] Take the product powder (over three sieve) (batch number: 010091-2306001) appropriate amount, take about 1.0g, put in the conical flask with plug, respectively, add 10% methanol 10ml, 25ml, 50ml, tightly plug, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool, shake, filter, take the filter solution, namely. See Figure 18 .

[0193] The results show that when the solvent addition amount is 25ml, the characteristic spectrum peak area is moderate. Therefore, the solvent addition amount of test sample is determined to be 25ml.

[0194] 2.2.5 Determine the test sample preparation method

[0195] Take the powder (over three sieves) of the product, take about 1.0 g, put in a conical flask with a plug, add 10% methanol solution 25 ml, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake, filter, take the filtrate, and get.

[0196] 2.3 Methodology Investigation

[0197] 2.3.1 Chromatographic Peak Identification

[0198] Preparation of Test Solution: According to the experimental conditions proposed above, prepare the test solution of hedgehog skin (hedgehog) medicinal material.

[0199] Preparation of Reference Solution: Take the appropriate amount of uracil, guanine, hypoxanthine, xanthine, inosine, guanosine reference substance, accurately weigh, add 10% methanol to prepare a solution containing 40 μg per 1 ml, and get.

[0200] Preparation of Boiled Hedgehog Skin Control Medicinal Material Solution: Take about 1.0 g of hedgehog skin (hedgehog) control medicinal material, put it in a conical flask with a plug, add 10% methanol 25 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake, filter, take the filtrate, and get the control medicinal material reference solution.

[0201] Preparation of Negative Control Solution: According to the experimental conditions proposed above, prepare the negative control solution of hedgehog skin (hedgehog) medicinal material.

[0202] Position the characteristic peaks of hedgehog skin (hedgehog) medicinal material. See Figure 19 .

[0203] Based on the above, 8 characteristic peaks in the medicinal material are included in the subsequent investigation, and the methodology is studied.

[0204] 2.3.2 Precision Test

[0205] Take the test solution of hedgehog skin (hedgehog) medicinal material, according to the proposed experimental method, continuously sample 6 times, 10 μl each time, calculate the retention time of each characteristic peak. See Table 8.

[0206] Table 8 Precision Investigation - Characteristic Peak Retention Time

[0207]

[0208] The results show that the precision of each characteristic peak retention time RSD is 0.05%-0.41%. The instrument precision is good.

[0209] 2.3.3 Reproducibility Investigation

[0210] Precisely weigh Echinocystis axungana (Miq.) Diels (batch number: 010091-2306001) 6 parts, and prepare and determine according to the proposed experimental method. See Table 9.

[0211] Table 9 Reproducibility Investigation - Relative Retention Time of Characteristic Peaks

[0212]

[0213]

[0214] The results show that the relative retention RSD of each characteristic peak is 0.00%-1.56%, and the method has good reproducibility.

[0215] 2.3.4 Intermediate Precision Investigation

[0216] On the basis of the above proposed experimental conditions, 12 parts of Echinocystis axungana (Miq.) Diels medicinal materials were precisely weighed to prepare the test sample solution, which was determined on high performance liquid chromatograph 1 and 2, respectively. See Table 10. Figure 20

[0217] Table 10 Intermediate Precision - Relative Retention Time Ratio

[0218]

[0219] The results show that the method has good intermediate precision.

[0220] 2.3.5 Durability Investigation

[0221] 2.3.5.1 Column Durability Investigation

[0222] On the basis of the above proposed experimental conditions, different brands of chromatographic columns 1, 2 and 3 were investigated, respectively. Figure 21 See Table 11.

[0223] Table 11 Column Durability Investigation - Relative Retention Time of Characteristic Peaks

[0224]

[0225] The results show that when different chromatographic columns are used, the relative retention time RSD values differ greatly. It is recommended to use column 1.

[0226] 2.3.5.2 Stability Investigation

[0227] On the basis of the above proposed experimental conditions, the same test sample solution was taken and determined at 0 h, 4 h, 8 h, 12 h, 16 h and 24 h, respectively. See Table 12.

[0228] Table 12 24-Hour Stability Investigation - Retention Time of Characteristic Peaks ​

[0229]

[0230] The results show that the RSD of the corresponding characteristic peak retention time is 0.27% to 1.56%, and the sample solution is relatively stable within 24 hours.

[0231] In summary, the RSD of the retention time / relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good.

[0232] 2.4 Determination of characteristic peaks and establishment of reference chromatogram

[0233] 2.4.1 Verification results of 16 batches of hedgehog skin (Erinaceus) medicinal materials

[0234] The proposed method was used to determine the characteristic chromatogram of 16 batches of samples, and the relative retention time was calculated. See Table 13. Figure 22 Figure 22 Among them, each chromatogram represents batch number S1: XXLS202305729; S2: XXLS202305730; S3: XXLS202305731; S4: XXLS202305732; S5: XXLS202305733; S6: XXLS202305734; S7: XXLS202305734; S8: XXLS202305736; S9: XXLS202305737; S10: XXLS202305738; S11: XXLS202305739; S12: XXLS202305740; S13: XXLS202305741; S14: XXLS202305742; S15: XXLS202305743; S16: 010091-2306001.

[0235] Table 13 Relative retention times of 16 batches of hedgehog skin (Erinaceus) medicinal materials

[0236]

[0237] According to the principles of stable relative retention time and detectable peaks in each batch of samples, a total of 8 peaks with good reproducibility were selected as characteristic peaks.

[0238] 2.4.2 Establishment of relative retention time limit

[0239] The results of the methodological investigations and verification are shown in Table 14:

[0240] Table 14 Summary of RSD% of each item in the method

[0241] ​

[0242] The results showed that, in addition to the column durability, the RSD values of the retention time or relative retention time of peaks 1 to 6 were small, basically meeting the requirements; the relative retention time difference of peaks 7 and 8 was large during the intermediate precision investigation, and subsequent consideration was given to using reference substances for identification.

[0243] The final provisions are as follows: 8 characteristic peaks should be present in the test substance chromatogram, and should correspond to the retention time of the 8 characteristic peaks in the reference substance chromatogram of the control medicinal material, among which peaks 3, 7, and 8 should correspond to the retention time of the corresponding reference substance peaks of the control substance, and the peak corresponding to the guanine control substance is the S peak. The relative retention times of peaks 1, 2, 4, 5, and 6 to the S peak should be within ±10% of the specified value, and the specified values are: 0.54 (peak 1), 0.75 (peak 2), 1.32 (peak 4), 1.46 (peak 5), and 1.67 (peak 6).

[0244] The characteristic chromatogram of 16 batches of hedgehog skin (Erinaceus europaeus) medicinal materials was synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and a control chromatogram of the characteristic chromatogram of hedgehog skin medicinal materials was established. See Figure 23 .

[0245] 2.5 Characteristic chromatogram verification of scalded hedgehog skin (Erinaceus europaeus) decoction pieces

[0246] Prepare the test substance solution of 16 batches of decoction pieces according to the results of "2.2 Preparation of test substance solution", and inject for determination and analysis according to the results of "2.1 Chromatographic conditions and system suitability test", and calculate the relative retention time. The results are shown in Figure 24 , Table 15. Figure 24 Among them, the batch numbers represented by each chromatogram are as follows: S1: TCWP-230601; S2: TCWP-230602; S3: TCWP-230603; S4: TCWP-230604; S5: TCWP-230605; S6: TCWP-230606; S7: TCWP-230607; S8: TCWP-230608; S9: TCWP-230609; S10: TCWP-230610; S11: TCWP-230611; S12: TCWP-230612; S13: TCWP-230613; S14: TCWP-230614; S15: TCWP-230615; S16: TCWP-230616.

[0247] Table 15 Relative retention times of characteristic peaks of 16 batches of scalded hedgehog skin decoction pieces

[0248]

[0249]

[0250] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peaks, a total of 8 peaks with good repeatability were selected as characteristic peaks.

[0251] The final specification is as follows: the chromatogram of the test sample should show 8 characteristic peaks, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 3, 7, and 8 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the guanine reference peak is the S peak. The relative retention times of peaks 1, 2, 4, 5, and 6 with the S peak should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.54 (peak 1), 0.75 (peak 2), 1.32 (peak 4), 1.46 (peak 5), and 1.67 (peak 6).

[0252] The characteristic chromatograms of 16 batches of scalded hedgehog skin slices were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and a reference chromatogram for the characteristic chromatograms of scalded hedgehog skin slices was established. See [link to relevant documentation]. Figure 25 .

[0253] 2.6 Method for determining the characteristic atlas of hedgehog skin (hedgehog) medicinal materials and processed slices

[0254] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile was used as mobile phase A, and 0.05% acetic acid solution was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 0.5 mL / min; the column temperature was 20 °C; and the detection wavelength was 250 nm. The theoretical plate number, calculated based on the guanine peak, should be no less than 5000.

[0255]

[0256] Preparation of reference solutions: Take 1g of hedgehog skin (hedgehog) reference material, place it in a stoppered conical flask, add 25ml of 10% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of guanine, inosine, and guanosine reference standards, accurately weigh them, and add 10% methanol to prepare solutions containing 40μg per ml, as reference solutions.

[0257] Preparation of the test solution: Weigh approximately 1.0 g of the powder (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, accurately add 25 ml of 10% methanol, seal tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 10% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0258] Determination method: precisely pipet 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

[0259] Example 3 HPLC characteristic chromatogram of scalded hedgehog skin (hedgehog) formula granules

[0260] 3.1 Chromatographic conditions and system suitability test The chromatographic conditions and system suitability test were as follows: octadecylsilane-bonded silica gel as the filler; acetonitrile as the mobile phase A, and 0.05% acetic acid solution as the mobile phase B, gradient elution was performed according to the following table; the detection wavelength was 250 nm. The theoretical plate number calculated according to the guanine peak should not be less than 5000.

[0261]

[0262] The reference solution was prepared as follows: 1 g of hedgehog skin (hedgehog) control medicinal material was placed in a conical flask with a stopper, 10% methanol 25 ml was added, and ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. After cooling, shaking and filtration, the filtrate was taken as the control medicinal material reference solution. In addition, guanine, inosine and guanosine reference substances were precisely weighed, 10% methanol was added to prepare a solution containing 40 μg per 1 ml, which was taken as the reference substance reference solution.

[0263] The test solution was prepared as follows: an appropriate amount of the product was finely ground, about 0.5 g was precisely weighed, placed in a conical flask with a stopper, 10% methanol 25 ml was precisely added, tightly stoppered, weighed, ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes, cooled, weighed again, the lost weight was made up with 10% methanol, shaken, filtered, and the filtrate was taken.

[0264] Determination method: precisely pipet 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

[0265] 3.2 Chromatographic conditions and system suitability test

[0266] 3.2.1 Wavelength selection

[0267] Based on the above proposed experimental conditions, full-wave band scanning was performed on the solutions of uracil, guanine, hypoxanthine, xanthine, inosine and guanosine using a diode array detector, and the chromatograms of the test solution at 210 nm, 230 nm, 250 nm, 270 nm, 290 nm and 310 nm were extracted, as shown in Figures 26-32 .

[0268] The results showed that when the detection wavelength was 250 nm, the amount of chromatographic peak information was larger, and the baseline of the chromatogram was more stable, so the detection wavelength was determined to be 250 nm.

[0269] 3.2.2 Column temperature investigation

[0270] Based on the above experimental conditions, the column temperature was investigated at 20℃, 25℃, 30℃ and 35℃, respectively. See Figure 33 .

[0271] The column temperature investigation results show that at different column temperatures, the column temperature is 20℃, the separation degree is good, the peak shape of the chromatogram is relatively symmetrical and the retention time is appropriate, so the column temperature is selected as 20℃.

[0272] 3.2.3 Flow rate investigation

[0273] Based on the above experimental conditions, the flow rate was investigated at 0.5ml / min, 0.6ml / min and 0.7ml / min, respectively. See Figure 34 .

[0274] The flow rate investigation results show that at different flow rates, the flow rate is 0.5ml / min, the separation degree is good, the peak shape of the chromatogram is relatively symmetrical and the retention time is appropriate, so the flow rate is selected as 0.5ml / min.

[0275] In summary, the chromatographic conditions and system suitability test of scalded hedgehog skin formula granules characteristic map are as follows: octadecylsilane bonded silica gel is used as the filler; acetonitrile is used as the mobile phase A, and 0.05% acetic acid solution is used as the mobile phase B, which is gradient eluted according to the following table; the flow rate is 1.0ml per minute; the column temperature is 25℃; and the detection wavelength is 246nm. The theoretical plate number calculated by alanine peak should not be less than 5000.

[0276]

[0277] 3.3 Preparation of test solution

[0278] 3.3.1 Investigation of extraction solvent

[0279] Take the product (batch number: 1903019) in an appropriate amount, grind it finely, take about 0.5g, put it in a conical flask with a plug, respectively add water, 10% methanol, 30% methanol, 70% methanol, methanol and 10% ethanol each 25ml, tightly plug, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool down, shake evenly, filter, take the filtrate, that is, the test solution. See Figure 35 .

[0280] The results show that when 10% methanol is used as the extraction solvent, the chromatographic peak information is larger and the peak shape is better, so the extraction solvent of the test sample is determined as 10% methanol.

[0281] 3.3.2 Investigation of extraction method

[0282] Take the product (batch number: 1903019) appropriate amount, grind, take about 0.5g, placed in a conical flask with plug, add 10% methanol 25ml, tight plug, respectively, for the test solution extraction method is reflux, ultrasonic (power 600W, frequency 40kHz) for investigation, extraction time 30 minutes, cool, shake, filter, take the filter solution, that is. See Figure 36 .

[0283] The results show that, in ultrasonic extraction and reflux extraction, the chromatogram effect is basically the same. This experiment selects ultrasonic extraction as the test solution extraction method.

[0284] 3.3.3 Extraction time investigation

[0285] Take the product (batch number: 1903019) appropriate amount, grind, take about 0.5g, placed in a conical flask with plug, add 10% methanol 25ml, tight plug, respectively, for the test solution ultrasonic treatment (power 600W, frequency 40kHz) 20 minutes, 30 minutes, 40 minutes, investigation, cool, shake, filter, take the filter solution, that is. See Figure 37 .

[0286] The results show that, in different extraction time conditions, the chromatogram effect is basically the same. In order to guarantee the extraction, the test solution extraction time is determined to be 30 minutes.

[0287] 3.3.4 Solvent addition amount investigation

[0288] Take the product (batch number: 1903019) appropriate amount, grind, take about 0.5g, placed in a conical flask with plug, respectively, add 10% methanol 10ml, 25ml, 50ml, tight plug, ultrasonic treatment (power 600W, frequency 40kHz) 30 minutes, cool, shake, filter, take the filter solution, that is. See Figure 38 .

[0289] The results show that, when the solvent addition amount is 25ml, the characteristic map chromatographic peak area is moderate. Therefore, the solvent addition amount of the test solution is determined to be 25ml.

[0290] In summary, the preparation method of the characteristic map test solution of the scalded hedgehog skin (hedgehog) formula granules is determined as follows: take the product appropriate amount, grind, take about 0.5g, placed in a conical flask with plug, add 10% methanol solution 25ml, tight plug, ultrasonic treatment (power 600W, frequency 40kHz) 30 minutes, cool, shake, filter, take the filter solution, that is.

[0291] 3.4 Methodology investigation

[0292] 3.4.1 Chromatographic peak identification

[0293] Preparation of Test Solution: The test solution of the Erzhihu skin (Hedgehog) formula granules was prepared according to the experimental conditions as above.

[0294] Preparation of Reference Solution: The uracil, guanine, guanosine, xanthine, inosine, guanosine reference substances were precisely weighed and added with 10% methanol to prepare a solution containing 40 μg per 1 ml, and the solution was obtained.

[0295] Preparation of Reference Solution of the Erzhihu Skin (Hedgehog) Reference Material: About 1.0 g of the Erzhihu skin (Hedgehog) reference material was placed in a conical flask with a stopper, 10% methanol 25 ml was added, and ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. After cooling, the solution was shaken and filtered. The filtrate was used as the reference solution of the reference material.

[0296] Preparation of Negative Control Solution: The negative control solution of the Erzhihu skin (Hedgehog) formula granules was prepared according to the experimental conditions as above.

[0297] The characteristic peaks of the Erzhihu skin (Hedgehog) formula granules were located. See Figure 39 .

[0298] 3.4.2 Precision Test

[0299] The test solution of the Erzhihu skin formula granules was taken, and 10 μl of the sample was continuously injected 6 times according to the experimental method. The retention time of each characteristic peak was calculated. See Table 16.

[0300] Table 16 Precision Investigation - Retention Time of Characteristic Peaks

[0301]

[0302] The results showed that the RSD value of the retention time of each characteristic peak was 0.01% to 0.32%. The precision of the instrument was good.

[0303] 3.4.3 Reproducibility Investigation

[0304] The Erzhihu skin formula granules were precisely weighed, and the preparation and determination were performed according to the experimental method. See Table 17.

[0305] Table 17 Reproducibility Investigation - Relative Retention Time of Characteristic Peaks

[0306]

[0307] The results showed that the RSD value of the relative retention time of each characteristic peak was 0.00% to 1.04%, and the results showed that the method had good reproducibility.

[0308] 3.4.4 Intermediate Precision Investigation

[0309] On the basis of the above experimental conditions, 12 parts of the prepared test sample solution were precisely weighed, and determined on HPLC 1 and 2, respectively, as shown in Table 18. Figure 40

[0310] Table 18 Intermediate precision - relative retention time ratio

[0311]

[0312] The results show that the intermediate precision of the method is good.

[0313] 3.4.5 Durability Investigation

[0314] 3.4.5.1 Investigation of Column Durability

[0315] On the basis of the above experimental conditions, different brands of chromatographic columns 1, 2 and 3 were investigated, respectively. See Table 19, Figure 41

[0316] Table 19 Investigation of column durability - relative retention time of characteristic peaks

[0317]

[0318] The results show that when different chromatographic columns are used, the RSD values of the relative retention time of the characteristic peaks are quite different. It is recommended to use column 1.

[0319] 3.4.5.2 Stability Investigation

[0320] On the basis of the above experimental conditions, the same test sample solution was taken and determined at 0 h, 4 h, 8 h, 12 h, 16 h and 24 h, respectively. See Table 20.

[0321] Table 20 Stability investigation - retention time of characteristic peaks

[0322]

[0323]

[0324] The results show that the RSD values of the corresponding characteristic peaks are 0.15% to 0.95%, and the sample solution is relatively stable within 24 hours.

[0325] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 8 characteristic peaks are included in the subsequent investigation.

[0326] 3.5 Determination of Characteristic Peaks and Establishment of Control Chromatogram

[0327] 3.5.1 Verification Results of Batch of Prepared Porcupine Skin (Porcupine) Formula Granules​​

[0328] The proposed method was used to determine the characteristic map of 3 batches of samples, and the relative retention time was calculated. See Figure 42 , Table 21.

[0329] Table 21 Relative retention time of 3 batches of scalded hedgehog skin (hedgehog) formula granules

[0330]

[0331] According to the principle of stable relative retention time and detectable peaks in each batch of samples, a total of 8 peaks with good reproducibility were selected as characteristic peaks.

[0332] 3.5.2 Establishment of relative retention time limit

[0333] The results of each investigation item and validation of the methodology are shown in Table 22:

[0334] Table 22 Summary of RSD% of each item of the methodology Relative retention time

[0335]

[0336] The test sample chromatogram should show 8 characteristic peaks, which should correspond to the relative retention time of the 8 characteristic peaks in the reference material chromatogram of the control drug, among which peaks 3, 7 and 8 should correspond to the relative retention time of the control reference peaks. The peak corresponding to the guanine control reference is S peak. The relative retention time of peaks 1, 2, 4, 5, 6 and S peak should be within ±10% of the specified value, which is 0.54 (peak 1), 0.75 (peak 2), 1.32 (peak 4), 1.46 (peak 5), and 1.67 (peak 6).

[0337] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprint (2012 edition) was used to synthesize the characteristic chromatogram of 3 batches of scalded hedgehog skin formula granules, and the control chromatogram of scalded hedgehog skin formula granules was established. See Figure 43 .

[0338] 3.6 Determination of characteristic chromatogram method of scalded hedgehog skin (hedgehog) formula granules

[0339] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, 0.05% acetic acid solution as mobile phase B, gradient elution according to the following table; flow rate was 0.5 ml per minute; column temperature was 20℃; detection wavelength was 250 nm. The theoretical plate number calculated by guanine peak should not be less than 5000.

[0340]

[0341] Reference solution preparation: Take 1 g of hedgehog skin (hedgehog) control medicinal material, place it in a conical flask with a stopper, add 10% methanol 25 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the filtrate, as the control medicinal material reference solution. Take appropriate amount of guanine, inosine, guanosine control, accurately weigh, add 10% methanol to make a solution containing 40 μg per 1 ml, as the reference solution of the control.

[0342] Test solution preparation: Take appropriate amount of the product, grind finely, take about 0.5 g, accurately weigh, place it in a conical flask with a stopper, accurately add 10% methanol 25 ml, tightly stop, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with 10% methanol, shake well, filter, take the filtrate, and get it.

[0343] Determination method: accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, determine, and get it.

[0344] The above is only the preferred embodiment of the present application, it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for constructing HPLC characteristic chromatogram of hedgehog skin medicinal material, decoction piece, standard decoction and its formula granules, comprising the following steps: A) hedgehog skin raw material is added to 10% methanol and ultrasonically treated, cooled, shaken, filtered, and the filtrate is taken to obtain a test solution; B) the test solution is determined by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of the hedgehog skin raw material; The high performance liquid chromatography chromatographic conditions are: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, and the mobile phase B is 0.05% acetic acid, gradient elution; The gradient elution is specifically as follows: 0~20min, A phase: 0%, B phase: 100%; 20~21min, A phase: 0%~1%, B phase: 100%~99%; 21~35min, A phase: 1%, B phase: 99%; 35~38min, A phase: 1%~2.5%, B phase: 99%~97.5%; 38~70min, A phase: 2.5%, B phase: 97.5%.

2. The construction method of claim 1, wherein, It also includes preparation of reference solution of control and reference solution of control medicinal material; Uracil, guanine, hypoxanthine, xanthine, inosine and guanosine are taken, 10% methanol is added to dissolve to obtain the reference solution of control; The hedgehog skin control medicinal material is taken, 10% methanol is added to ultrasonically extract to obtain the reference solution of control medicinal material.

3. The construction method of claim 1, wherein, The chromatographic column is a C18 column with a specification of 5μm, 4.6*250mm; the column temperature is 20℃.

4. The construction method of claim 1, wherein, The flow rate of the mobile phase is 0.5mL / min; the detection wavelength is 250nm; the injection amount is 10μL.

5. The construction method of claim 1, wherein, The similarity of the HPLC characteristic chromatogram of the hedgehog skin medicinal material, decoction piece, standard decoction and its formula granules is evaluated by using traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the HPLC standard characteristic chromatogram of the hedgehog skin medicinal material, decoction piece, standard decoction and its formula granules composed of 8 characteristic peaks, wherein peak 2 is the uracil peak; peak 3 is the guanine peak; peak 5 is the hypoxanthine peak; peak 6 is the xanthine peak; peak 7 is the inosine peak; peak 8 is the guanosine peak; Guanine is taken as the reference peak S peak, wherein peak 3, peak 7 and peak 8 should correspond to the retention time of the corresponding control reference peak.

6. The construction method according to claim 5, characterized in that, In the HPLC characteristic chromatogram of the hedgehog skin medicinal material, decoction piece, standard decoction and its formula granules, guanine is taken as the reference peak S peak, the relative retention time of peak 1, peak 2, peak 4, peak 5, peak 6 and S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: 0.54 (peak 1), 0.75 (peak 2), 1.32 (peak 4), 1.46 (peak 5), 1.67 (peak 6).

7. The construction method of claim 1, wherein, In step A), the ultrasonic treatment power is 600W, and the frequency is 40kHz; the extraction time is 20~40min.

8. The construction method of claim 1, wherein, The hedgehog skin raw material is a hedgehog skin medicinal material, decoction piece, standard decoction and its formula granules.

9. The construction method of claim 7, wherein, The mass to volume ratio of the hedgehog skin standard decoction and the hedgehog skin formula granules to the solvent is 0.5g:25mL; The mass to volume ratio of the hedgehog skin medicinal material and decoction piece to the solvent is 1g:25mL.

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