A method for determining the content of sandalwood medicinal material, decoction piece, standard decoction and formula granule

Abstract

The application provides a method for determining the content of sandalwood medicinal materials, decoction pieces, standard decoction and formula granules, which takes verbascoside as a control sample, and detects the test sample solution and the control sample solution through liquid chromatography, and takes acetonitrile-water with a volume ratio of (15-20):(80-85) as a mobile phase. The method can scientifically identify sandalwood medicinal materials, decoction pieces, standard decoction and formula granules, can control the internal quality of sandalwood and related preparations from the whole and macro, and can effectively guarantee the curative effect of the medicine, so that the medicinal materials and related preparations can be more regularly controlled in quality.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug detection, and particularly relates to a determination method for the content of sandalwood medicinal materials, decoction pieces, standard decoctions and formula granules. BACKGROUND

[0002] Sandalwood has a long history of use in medicine and was first recorded in the Han Dynasty herbal work Shanghan Zabing, which listed sandalwood as Xiaoping and recorded it as 'purple true sandalwood'. However, sandalwood is now a collective term for plants in the genus Santalum, and is not the sandalwood used today. In Shanghan Zabing, it is recorded that'sandalwood has a salty taste and is slightly cold. It is used to treat evil toxins and wind toxins.' This is different from the modern Pharmacopoeia of the People's Republic of China, which records that'sandalwood warms the middle and relieves pain. It is used for cold stagnation of qi, chest and diaphragm discomfort, chest pain, stomach and abdominal pain, vomiting and loss of appetite.' In Ben Cao Gang Mu, it is recorded that 'white vajra sandalwood is pungent, warm and non-toxic.' 'Purple sandalwood is salty, slightly cold and non-toxic.' This is ambiguous, and the record of purple sandalwood is consistent with that in Shanghan Zabing, and the record of white vajra sandalwood is consistent with the nature and taste in the 2020 edition of the Pharmacopoeia of the People's Republic of China. In Ben Cao Cong Xin, it is recorded that 'white sandalwood is pungent and warm. It regulates the spleen and lungs, benefits the chest and diaphragm, treats vomiting due to dysphagia, stops abdominal pain, repels ghosts and kills insects, and promotes appetite and food intake.' This is also basically consistent with the nature, taste and function and indications in the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0003] Current research has found that the use of sandalwood mixed with purple sandalwood and yellow sandalwood does exist. There is little research on the determination of the content of sandalwood. In order to evaluate and control the quality of sandalwood as a whole, it is urgent to establish a unified method for determining the content of sandalwood. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a determination method for the content of sandalwood medicinal materials, decoction pieces, standard decoctions and formula granules.

[0005] To achieve this purpose, the present application adopts the following technical solutions:

[0006] The present application provides a determination method for the content of sandalwood medicinal materials, decoction pieces, standard decoctions and formula granules, comprising:

[0007] The control solution and the test solution are determined by liquid chromatography, and the determination is completed.

[0008] The control in the control solution is verbascoside.

[0009] The conditions of the liquid chromatography are as follows: acetonitrile-water is used as the mobile phase, and the volume ratio of acetonitrile to water in the acetonitrile-water is (15-20):(80-85).

[0010] More preferably, the volume ratio of acetonitrile to water in the acetonitrile-water is (17-19):(81-83).

[0011] Preferably, the test sample solution is extracted after mixing the test sample with 30-80 vol% methanol.

[0012] Preferably, the test sample solution is extracted after mixing the test sample with 50 vol% methanol.

[0013] Preferably, the mass-volume ratio of the test sample to 30-80 vol% methanol is 1:(100-500).

[0014] Preferably, the mass-volume ratio of the test sample to 50 vol% methanol is 1:(100-500).

[0015] Preferably, the extraction time is not less than 30 min.

[0016] Preferably, the control sample solution is extracted after mixing the verbascoside with 30-80 vol% methanol.

[0017] Preferably, the concentration of verbascoside in the control sample solution is 1-384 μg / mL.

[0018] Preferably, the amount of the control sample solution and the test sample solution added is independently selected from 8-12 μL.

[0019] More preferably, the amount of the control sample solution and the test sample solution added is both 10 μL.

[0020] Preferably, the filler of the chromatographic column in the liquid chromatography is octadecylsilane-bonded silica gel.

[0021] Preferably, the theoretical plate number of the chromatographic column calculated according to the peak of verbascoside is not less than 3000.

[0022] Preferably, the detection wavelength of the liquid chromatography is 300-340 nm.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application provides a method for determining the content of sandalwood medicinal materials, decoction pieces, standard decoctions and formula granules, which uses verbascoside as a control sample and detects the test sample solution and the control sample solution by liquid chromatography.

[0025] It is verified that the method can effectively separate and determine the verbascoside component of sandalwood medicinal materials, decoction pieces, standard decoction and formula granules, can effectively ensure the uniformity and stability of the quality of sandalwood medicinal materials, decoction pieces, standard decoction and formula granules, and provide guidance for the quality control of sandalwood. Moreover, the method provided by the application is comprehensive in detection, simple in operation, good in stability, high in precision, good in repeatability, and easy to master. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the ultraviolet absorption spectrum of verbascoside;

[0027] Figure 2 is the investigation graph of sandalwood formula granules as the test sample in different mobile phases;

[0028] Figure 3 is the chromatogram obtained by carrying out specificity investigation of sandalwood formula granules as the test sample;

[0029] Figure 4 is the standard curve graph of verbascoside. DETAILED DESCRIPTION

[0030] The technical solutions of the application will be described clearly and completely in combination with the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0031] In view of the problem that there is no unified sandalwood content determination method in the prior art, the application provides a sandalwood medicinal material, decoction piece, standard decoction and formula granule content determination method, comprising:

[0032] The control solution and the test solution are determined by liquid chromatography, and the determination is completed.

[0033] The control in the control solution is verbascoside.

[0034] In the application, the test solution is the solution of sandalwood medicinal materials, decoction pieces, standard decoction and formula granules, that is, sandalwood medicinal materials, decoction pieces, standard decoction and formula granules are test samples.

[0035] In the application, the preparation method of different test samples is explored and established. The exploration factors mainly include the selection of extraction solvent, the investigation of extraction time and the investigation of the addition amount of extraction solvent in the preparation process of different test sample solutions.

[0036] In some embodiments of the present application, the test sample solution can be obtained by mixing the test sample with 30-80 vol% methanol, preferably 50 vol% methanol, and then extracting. In the present application, the extraction method can be reflux extraction or ultrasonic extraction, and is not particularly limited. In order to ensure the completeness of the extracted active ingredients, the reflux extraction or ultrasonic extraction time is preferably not less than 30 min. When the amount of the test sample is 1 g, the amount of the extraction solvent can be 100-500 mL, which can be 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL or 500 mL, etc.

[0037] It should be noted that when the extraction method is ultrasonic extraction, the power of the ultrasonic extraction in the present application is preferably 500-700 W, more preferably 600 W, and the frequency is preferably 30-50 kHz, more preferably 40 kHz.

[0038] In some embodiments of the present application, when the test sample is sandalwood formula granules, the test sample solution is prepared according to the following method:

[0039] Take the product (i.e., sandalwood formula granules) in an appropriate amount, grind finely, take about 0.1 g, accurately weigh, place in a conical flask with a stopper, accurately add 50 vol% methanol 50 mL, tightly seal, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, cool, re-weigh, make up the weight loss with 50 vol% methanol, shake well, filter, and take the filtrate, which is obtained.

[0040] In some embodiments of the present application, when the test sample is sandalwood medicinal material, the test sample solution is prepared according to the following method:

[0041] Take the product, (pass through a No. 3 sieve) about 0.5 g, accurately weigh, place in a conical flask with a stopper, accurately add 50 vol% methanol solution 50 mL, tightly seal, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, cool, re-weigh, make up the weight loss with 50 vol% methanol solution, shake well, filter, and take the filtrate, which is obtained.

[0042] In some embodiments of the present application, when the test sample is sandalwood decoction pieces, the test sample solution is prepared according to the following method:

[0043] Take the product, (pass through a No. 3 sieve) about 0.5 g, accurately weigh, place in a conical flask with a stopper, accurately add 50 vol% methanol solution 50 mL, tightly seal, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, cool, re-weigh, make up the weight loss with 50 vol% methanol solution, shake well, filter, and take the filtrate, which is obtained.

[0044] In some embodiments of the present application, when the test sample is a sandalwood standard decoction, the test sample solution is prepared as follows:

[0045] About 0.1 g of the product powder is precisely weighed, placed in a conical flask with a stopper, 50 mL of 50 vol% methanol solution is precisely added, tightly stoppered, weighed, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 min, allowed to cool, weighed again, the lost weight is made up with 50 vol% methanol solution, shaken well, filtered, and the filtrate is obtained.

[0046] In the present application, the control in the control solution is verbascoside. The compounds currently isolated from sandalwood medicinal materials are mainly sesquiterpenes. In addition, there are various chemical components such as monoterpenes and lignans. The sesquiterpene components and monoterpenes are lipophilic components. Most of the current research is limited to lipophilic components. In the present experiment, 50 vol% methanol is used to study both hydrophilic and lipophilic components. Through the development of chromatographic conditions, verbascoside is identified, which belongs to phenylethanoid glycosides and is a slightly hydrophilic compound with a relatively high content. Therefore, through screening, verbascoside is specifically selected as the content determination index of sandalwood, which can better exploit the medicinal value of sandalwood.

[0047] In some embodiments of the present application, the control solution is obtained by mixing verbascoside with 30-80 vol% methanol, preferably 50 vol% methanol. The extraction method can refer to the preparation of the test sample solution described above.

[0048] Through linear investigation, the concentration of verbascoside in the preferred control solution of the present application is preferably within 1-384 μg / mL, and more preferably within 5-300 μg / mL. The linear curve of verbascoside obtained in this way can show a good linear relationship.

[0049] After obtaining the test sample solution and the control solution according to the above method, the present application determines the control solution and the test sample solution by liquid chromatography. It should be noted that not all mobile phases involved in liquid chromatography can obtain better detection results. In the present application, different mobile phases are used for research. The results show that acetonitrile-water under isocratic elution conditions can obtain a relatively complete chromatographic peak type, and is friendly to the chromatographic column. Therefore, acetonitrile-water is used as the mobile phase of the chromatographic determination method. The volume ratio of acetonitrile to water in the acetonitrile-water is preferably (15-20):(80-85), more preferably (17-19):(81-83), and most preferably 19:81.

[0050] The application determines the optimal inspection wavelength of the content determination of verbascoside by full wavelength spectrum collection of verbascoside control solution and analysis of the spectrum, preferably 300-340nm, and more preferably 330nm.

[0051] The liquid chromatography condition established by the liquid chromatography condition and system adaptability test is as follows:

[0052] Octadecylsilane-bonded silica gel is used as the filler (the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5um);acetonitrile-water solution (the volume ratio of acetonitrile and water is (15-20):(80-85)) is used as the mobile phase;the detection wavelength is 300-340nm. The theoretical plate number calculated according to the verbascoside peak should be not less than 3000.

[0053] In the application, the specific determination method is as follows:

[0054] Precisely take 8-12ul, preferably 10ul of the control solution and the test solution, inject into the liquid chromatograph, and determine according to the above liquid chromatography condition.

[0055] After determining the preparation method of the test solution and the determination method of the liquid chromatography, the application also investigates the determination method of the liquid chromatography, mainly including the specificity experiment, the precision investigation, the repeatability investigation, the intermediate precision investigation (different instrument investigation, different personnel time investigation), the linearity investigation, the stability investigation, the accuracy investigation, the durability investigation, etc. The results show that the determination method provided by the application has good specificity, precision, repeatability, intermediate precision, linearity, stability, accuracy and durability.

[0056] In the specificity investigation, the negative solution is detected by the liquid chromatography together with the control solution and the test solution. In the application, the negative solution is the solvent prepared after not adding the sample of Santalum album, and the addition amount is 8-12ul, preferably 10ul. The results show that the chromatogram of the negative solution has no interference on the determination of the measured peak, indicating that the method has good specificity.

[0057] Further, after the above investigation is completed, the determination method of the liquid chromatography is verified by a series of batches of test products. The results show that the method can effectively separate and determine the verbascoside component of Santalum album, decoction pieces, standard decoction and formula granules, effectively ensure the uniformity and stability of the quality of Santalum album, decoction pieces, standard decoction and formula granules, and provide guidance for the quality control of Santalum album.

[0058] In order to further illustrate the present application, the following examples are provided in detail. The experimental materials used in the following examples of the present application are all commercially available. The "%" involved below, unless otherwise specified, means volume percentage.

[0059] Example 1

[0060] 1 Experimental instruments and materials

[0061] Instrument 1 (Thermo), Instrument 2 (Agilent 1260), Instrument 3 (Shimadzu LC-20);

[0062] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0063] Ultrapure water machine: CelluSafe 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0064] Ultrasonic cleaner: KQ5200DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0065] Chromatographic column: Chromatographic column 1 (TC-C18), chromatographic column 2 (SB-C18), chromatographic column 3 (Extend-C18);

[0066] Acetonitrile, phosphoric acid, methanol, formic acid are chromatographically pure; water is ultrapure water, and other reagents are analytically pure;

[0067] Mucorin (Majorsin) control (China Food and Drug Inspection Research Institute, batch number: 111530-201914, content is 95.2%);

[0068] Santalum formula granules: TX-01, TX-02, TX-03, TX-04, TX-05, provided by Sichuan New Green Pharmaceutical Technology Development Co., Ltd.;

[0069] Santalum medicinal materials: TX-YC-01, TX-YC-02, TX-YC-03, TX-YC-04, TX-YC-05, provided by Sichuan New Green Pharmaceutical Technology Development Co., Ltd.;

[0070] Santalum decoction pieces: TX-YP-01, TX-YP-02, TX-YP-03, TX-YP-04, TX-YP-05, provided by Sichuan New Green Pharmaceutical Technology Development Co., Ltd.;

[0071] Santalum standard decoction: TX-BT-01, TX-BT-02, TX-BT-03, TX-BT-04, TX-BT-05, provided by Sichuan New Green Pharmaceutical Technology Development Co., Ltd.

[0072] 2 Method for determination of granule content of the formula

[0073] 2.1 Granule content of sandalwood formula

[0074] 2.1.1 Preparation of test solution

[0075] Take an appropriate amount of the product, finely grind, take about 0.1 g, accurately weigh, place in a conical flask with a stopper, accurately add 50% methanol 50 mL, tightly seal, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, cool, re-weigh, make up the weight loss with 50% methanol, shake well, filter, take the filtrate, and obtain.

[0076] 2.1.2 Preparation of reference solution

[0077] Take an appropriate amount of verbascoside reference substance, accurately weigh, add 50% methanol solution to prepare a solution containing 40 μg per 1 mL, and obtain.

[0078] 2.1.3 Preparation of negative solution

[0079] Take an appropriate amount of auxiliary material, take about 0.1 g, accurately weigh, place in a conical flask with a stopper, accurately add 50% methanol 50 mL, tightly seal, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, cool, re-weigh, make up the weight loss with 50% methanol, shake well, filter, take the filtrate, and obtain.

[0080] 2.2 Chromatographic conditions

[0081] Chromatographic conditions and system suitability test

[0082] Use octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); use acetonitrile-water (volume ratio 19:81) as the mobile phase; the detection wavelength is 330 nm. The theoretical plate number calculated according to the verbascoside peak should not be less than 3000.

[0083] Determination method: accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and obtain.

[0084] 2.2.1 Determination of detection wavelength

[0085] On the basis of the above proposed test conditions, full-wave band scanning was performed on verbascoside, and the results are shown in Figure 1 .

[0086] The results show that through the analysis of the spectrum, the optimal detection wavelength for the determination of verbascoside in sandalwood granules is 330 nm.

[0087] 2.2.2 Investigation of different mobile phases

[0088] Based on the above proposed experimental conditions, the mobile phase is acetonitrile-water (volume ratio of acetonitrile, water is 19:81), acetonitrile-0.1% formic acid (volume ratio of acetonitrile, 0.1% formic acid is 19:81), acetonitrile-0.1% phosphoric acid (volume ratio of acetonitrile, 0.1% phosphoric acid is 19:81), methanol-water (volume ratio of methanol, water is 19:81) are investigated, and the results are shown in Figure 2 .

[0089] The results show that the mobile phase of acetonitrile-water, acetonitrile-0.1% formic acid, acetonitrile-0.1% phosphoric acid can separate verbascoside well, and methanol-water cannot effectively separate. However, due to the presence of acidic substances in acetonitrile-0.1% formic acid and acetonitrile-0.1% phosphoric acid, which can damage the chromatographic column, therefore, the mobile phase of acetonitrile-water is preferred in the present application.

[0090] 2.3 Investigation of test sample preparation method

[0091] 2.3.1 Investigation of extraction method

[0092] Take the product, grind it finely, take about 0.1 g, accurately weigh, place it in a conical flask with a plug, accurately add 50% methanol 50 mL, tightly plug, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) or reflux extraction for 30 min, cool, weigh again, make up the weight loss with 50% methanol, shake well, filter, take the filtrate, and obtain. Suck each test sample solution into the liquid chromatograph, calculate the content of verbascoside after determination, and the results are shown in Table 1.

[0093] Table 1 Investigation of extraction method

[0094] Extraction method Verbascoside content (mg / g) Ultrasonic 10.69 Refiux 10.70

[0095] The results show that the content of verbascoside obtained by ultrasonic extraction and reflux extraction is not much different, and ultrasonic extraction is selected for subsequent investigation.

[0096] 2.3.2 Investigation of extraction solvent

[0097] Take the product, grind it finely, take about 0.1 g, accurately weigh, place it in a conical flask with a plug, accurately add 50% methanol, 80% methanol, 50% methanol, 30% methanol, water, each 50 mL, tightly plug, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the weight loss with the respective solvent, shake well, filter, take the filtrate, and obtain. Suck each test sample solution into the liquid chromatograph, calculate the content of verbascoside after determination, and the results are shown in Table 2.

[0098] Table 2 Investigation of extraction solvent

[0099] Extraction solvent Verbascoside content (mg / g) Methanol 4.87 80% methanol 8.54 50% methanol 10.69 30% methanol 8.14 10% methanol 5.62 Water 4.68

[0100] The results show that the content of verbascoside is the highest when 50% methanol is used as the solvent.

[0101] 2.3.3 Extraction time investigation

[0102] Take the product, grind finely, take about 0.1 g, accurately weigh, place in a conical flask with a stopper, accurately add 50% methanol 50 mL, tightly seal, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, 45 min, 60 min, cool, weigh again, make up the weight loss with 50% methanol, shake well, filter, take the filtrate, and obtain. Inject each test sample solution into the liquid chromatograph, calculate the content of verbascoside after determination, and the results are shown in Table 3.

[0103] Table 3 Extraction time investigation

[0104]

[0105] The results show that the extraction time has little effect on the extraction of verbascoside, and 30 min is selected as the subsequent investigation.

[0106] 2.3.4 Investigation of the amount of extraction solvent added

[0107] Take the product, grind finely, take about 0.1 g, accurately weigh, place in a conical flask with a stopper, accurately add 50% methanol 25 mL, 50 mL, 100 mL, tightly seal, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the weight loss with 50% methanol, shake well, filter, take the filtrate, and obtain. Inject each test sample solution into the liquid chromatograph, calculate the content of verbascoside after determination, and the results are shown in Table 4.

[0108] Table 4 Investigation of the amount of extraction solvent added

[0109] Solvent addition amount (mL) Verbascoside content (mg / g) 25 10.18 50 10.69 100 10.12

[0110] The results show that the content is the highest when the amount of solvent added is 50 mL, and therefore 50 mL is selected as the final amount of extraction solvent added.

[0111] 2.4 Methodology verification

[0112] 2.4.1 Specificity test

[0113] Preparation of test sample solution: prepare the test sample solution of sandalwood formula granules according to the above proposed experimental conditions.

[0114] Preparation of control solution: accurately weigh appropriate amount of control sample of verbascoside, add 50% methanol solution to prepare a solution containing 40 μg per 1 mL, and obtain the control solution.

[0115] Preparation of negative control solution: prepare the negative control solution without sandalwood granules according to the experimental conditions proposed above.

[0116] The detection results are shown in Table 3. Figure 3 The results show that the negative control solution has no interference with the determination of the peak to be measured, indicating that the method has good specificity.

[0117] 2.4.2 Precision test

[0118] Take the control solution and continuously inject 6 times, and record the peak area of verbascoside. The results are shown in Table 5.

[0119] Table 5 Precision test

[0120]

[0121]

[0122] The results show that the instrument has good precision.

[0123] 2.4.3 Reproducibility test

[0124] Accurately weigh 6 portions of sandalwood formula granules (batch number: TX-01) as test samples, prepare test sample solutions according to the experimental method proposed above and determine the content of verbascoside in the 6 test samples. The results are shown in Table 6.

[0125] Table 6 Reproducibility test

[0126] Serial number Verbascoside content (mg / g) 1 10.59 12 10.71 3 10.61 4 10.48 5 10.63 6 10.65

[0127] The results show that the method has good reproducibility.

[0128] 2.4.4 Recovery rate

[0129] Take about 0.05 g of test sample (batch number: TX-01, content of verbascoside 10.6 mg / g) for a total of 6 portions, accurately weigh, and accurately add verbascoside control stock solution, prepare test sample solutions according to the proposed method and determine the recovery rate, the results are shown in Table 7. The calculation formula is as follows (wherein, the determined amount is the content of verbascoside measured in the sample; the sample content is the content of verbascoside in 0.05 g of sample; the added amount is the content of verbascoside added to the sample before preparation) :

[0130]

[0131] Table 7 Experimental results of verbascoside sample loading recovery rate

[0132] Number Determination result (mg) Recovery rate (%) 1 0.86551 100.6 2 0.87339 99.6 3 0.87213 100.3 4 0.86986 100.2 5 0.87012 100.7

[0133] The results show that the method has good accuracy.

[0134] 2.4.5 Linear relationship

[0135] Take an appropriate amount of verbascoside, place it in a 25 mL volumetric flask, and dissolve it with 50% methanol to prepare a verbascoside solution containing 383.92256 μg / mL (purity 95.2%). Then dilute it to concentrations of 1.53569024 μg / mL, 7.6784512 μg / mL, 15.3569024 μg / mL, 38.392256 μg / mL, 115.176768 μg / mL, and 383.92256 μg / mL, respectively. Precisely take 10 μL of each solution, inject it into the liquid chromatograph, and obtain the peak area. Plot the response curve with the concentration (X, μg / mL) as the horizontal coordinate and the peak area (Y) as the vertical coordinate. The results are shown in Table 8, Figure 4 .

[0136] Table 8 Analysis results of verbascoside standard curve

[0137] Sample concentration (X) 1.53569024 7.6784512 15.3569024 38.392256 115.176768 383.92256 Peak area (Y) 23755 126518 255417 645012 1975828 6499529

[0138] The results show that when the verbascoside concentration range is 1.53569024-383.92256 μg / mL, the linear relationship is y = 16945x + 345.68, R 2 = 1. This indicates that the concentration range of 1.53569024-383.92256 μg / mL has a good linear relationship.

[0139] 2.4.6 Intermediate precision investigation

[0140] 2.4.6.1 Investigation of different instruments

[0141] Based on the above proposed experimental conditions, two portions of sandalwood formula granules (batch number: TX-01) were precisely weighed, and the test sample solutions were prepared. The content of verbascoside in the test sample solutions was calculated by measuring them on instrument 1, instrument 2, and instrument 3, respectively. The results are shown in Table 9.

[0142] Table 9 Investigation of different instruments

[0143]

[0144]

[0145] The results show that the different instruments have good robustness.

[0146] 2.4.6.2 Different personnel and time investigation

[0147] On the basis of the experimental conditions proposed above, two samples of sandalwood formula granules (batch number: TX-01) were precisely weighed by different personnel (A, B) at different times (T1, T2), respectively, to prepare the test sample and determine the content of verbascoside in the test sample solution. The results are shown in Table 10.

[0148] Table 10 Personnel and time investigation

[0149]

[0150] The results show that the intermediate precision of this method is good.

[0151] 2.4.7 Durability investigation

[0152] On the basis of the experimental conditions proposed above, the column 1, column 2 and column 3 were investigated respectively. The results are shown in Table 11.

[0153] Table 11 Investigation of column durability

[0154]

[0155]

[0156] The results show that the durability of this method is good.

[0157] 2.4.8 Stability investigation

[0158] On the basis of the experimental conditions proposed above, the same test sample solution was taken and the chromatographic peak area of verbascoside was determined at 0h, 5h, 10h, 15h, 20h and 24h, respectively. The results are shown in Table 12.

[0159] Table 12 Stability investigation

[0160] Ingredient 0h 5h 10h 15h 20h 24h Verbascoside 593202 596238 579999 581366 582598 567920

[0161] The results show that the sample solution is relatively stable within 24 hours.

[0162] 2.5 Validation results of 5 batches of sandalwood formula granules

[0163] The content of 5 batches of samples was determined by the proposed method. The results are shown in Table 13.

[0164] Table 13 Validation results of the content of 5 batches of sandalwood formula granules

[0165] Batch number Verbascoside content (mg / g) TX-01 7.4 TX-02 7.3 TX-03 7.0 TX-04 7.2 TX-05 7.1

[0166] The results show that the method can effectively detect sandalwood formula granules.

[0167] Example 2

[0168] 1 Experimental instruments and materials

[0169] Reference Example 1

[0170] Sandalwood medicinal materials: TX-YC-01, TX-YC-02, TX-YC-03, TX-YC-04, TX-YC-05.

[0171] 2 Establishment of medicinal material content determination method

[0172] 2.1 Sandalwood medicinal materials

[0173] 2.1.1 Preparation of test sample solution

[0174] Take about 0.5 g of the product (pass through a No. 3 sieve), accurately weigh, place in a conical flask with a stopper, accurately add 50% methanol solution 50 mL, tightly seal, weigh, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the weight loss with 50% methanol solution, shake well, filter, take the filtrate, and obtain.

[0175] 2.1.2 Preparation of reference substance solution

[0176] Reference Example 1.

[0177] 2.1.3 Preparation of negative solution

[0178] Reference Example 1.

[0179] 2.2 Chromatographic conditions

[0180] Use octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); use acetonitrile-water solution (volume ratio 19:81) as the mobile phase; detection wavelength is 330 nm. The theoretical plate number should not be less than 3000 calculated according to the peak of verbascoside.

[0181] Determination method: accurately pipette 10 μL of the reference substance solution and the test sample solution respectively, inject into the liquid chromatograph, and determine, and obtain.

[0182] 2.3 Methodology verification

[0183] Reference Example 1, methodology verification, the results show that the method has good specificity, precision, repeatability, accuracy, durability, intermediate precision and stability, and the standard curve of verbascoside shows good linear relationship.

[0184] 2.4 Verification results of 5 batches of sandalwood medicinal materials

[0185] The content of 5 batches of samples was determined by the proposed method. As shown in Table 14.

[0186] Table 14 5 batches of sandalwood medicinal materials verification

[0187] Serial number Batch number Verbascoside content (%) Whether qualified 1 TX-YC-01 0.31 Qualified 2 TX-YC-02 0.30 Qualified 3 TX-YC-03 0.33 Qualified 4 TX-YC-04 0.38 Qualified 5 TX-YC-05 0.70 Qualified

[0188] Example 3

[0189] 1 Experimental instruments and materials

[0190] Reference Example 1

[0191] Sandalwood decoction pieces: TX-YP-01, TX-YP-02, TX-YP-03, TX-YP-04, TX-YP-05.

[0192] 2 Decoction piece content determination method establishment

[0193] 2.1 Sandalwood decoction pieces

[0194] 2.1.1 Preparation of test sample solution

[0195] Take about 0.5 g of the product (pass through No. 3 sieve), accurately weigh, place in a conical flask with a plug, accurately add 50% methanol solution 50 mL, tightly plug, weigh, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the weight loss with 50% methanol solution, shake well, filter, take the filtrate, and you get it.

[0196] 2.1.2 Preparation of control solution

[0197] Reference Example 1.

[0198] 2.1.3 Preparation of negative solution

[0199] Reference Example 1.

[0200] 2.2 Chromatographic conditions

[0201] Octadecylsilane-bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile-water solution (volume ratio 19:81) as mobile phase; detection wavelength 330 nm. The theoretical plate number should not be less than 3000 calculated by the peak of verbascoside.

[0202] Determination method: accurately pipette 10 μL of the control solution and the test sample solution respectively, inject into the liquid chromatograph, determine, and you get it.

[0203] 2.3 Methodology verification

[0204] Reference Example 1 was carried out to verify the methodology, and the results showed that the method had good specificity, precision, repeatability, accuracy, robustness, intermediate precision, and stability, and the standard curve of verbascoside showed a good linear relationship.

[0205] 2.4 Verification results of 5 batches of sandalwood slices

[0206] The content of 5 batches of samples was determined by the proposed method. As shown in Table 15.

[0207] Table 15 Verification of 5 batches of sandalwood slices

[0208]

[0209]

[0210] Example 4

[0211] 1 Experimental instruments and materials

[0212] Reference Example 1

[0213] Sandalwood standard decoction: TX-BT-01, TX-BT-02, TX-BT-03, TX-BT-04, TX-BT-05.

[0214] 2 Establishment of standard decoction content determination method

[0215] 2.1 Sandalwood standard decoction

[0216] 2.1.1 Preparation of test solution

[0217] About 0.1 g of the product powder was accurately weighed, placed in a conical flask with a plug, 50 mL of 50% methanol solution was accurately added, tightly sealed, weighed, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 min, cooled, weighed again, the lost weight was made up with 50% methanol solution, shaken well, filtered, and the filtrate was taken, i.e. the test solution was obtained.

[0218] 2.1.2 Preparation of control solution

[0219] Reference Example 1.

[0220] 2.1.3 Preparation of negative solution

[0221] Reference Example 1.

[0222] 2.2 Chromatographic conditions

[0223] The octadecylsilane-bonded silica gel was used as the filler (the column length was 250 mm, the inner diameter was 4.6 mm, and the particle size was 5 μm); the acetonitrile-water solution (the volume ratio was 19:81) was used as the mobile phase; and the detection wavelength was 330 nm. The theoretical plate number should be no less than 3000 according to the peak of verbascoside.

[0224] Determination method: 10 μL of the control solution and the sample solution was respectively injected into the liquid chromatograph, and then determination was performed, and thus the content was obtained.

[0225] 2.3 Methodology verification

[0226] According to the reference example 1, the methodology verification was performed, and the results showed that the method had good specificity, precision, repeatability, accuracy, durability, intermediate precision and stability, and the standard curve of verbascoside had good linear relationship.

[0227] 2.4 Verification results of 5 batches of sandalwood standard decoction

[0228] The content of 5 batches of samples was determined by the prepared method. As shown in Table 12.

[0229] Table 1 Verification of 5 batches of sandalwood standard decoction

[0230]

[0231]

[0232] It should be noted that the chromatographic conditions and system suitability test, and the preparation method of the test sample were also performed for the test sample of sandalwood medicinal material, sandalwood standard decoction and sandalwood decoction pieces, and the obtained conclusion was consistent with the test sample of sandalwood formula granules. In order to avoid being complicated, the details are not described herein.

[0233] The above description of the disclosed embodiments enables one skilled in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for determining the content of sandalwood medicinal materials, processed slices, standard decoctions, and formulated granules, characterized in that, include: The reference solution and the test solution are determined by liquid chromatography. The reference standard in the reference solution is verbascoside; The conditions for the liquid chromatography are as follows: acetonitrile-water is used as the mobile phase, and the volume ratio of acetonitrile to water in the acetonitrile-water mixture is (17~19):(81~83); the detection wavelength is 300~340 nm. The concentration of verbascoside in the reference solution was 1~384 μg / mL; The test solution is obtained by mixing the test sample with 30-80 vol% methanol and then extracting it.

2. The determination method according to claim 1, characterized in that, The test solution was obtained by mixing the test sample with 50 vol% methanol and then extracting it.

3. The determination method according to claim 1, characterized in that, The mass-to-volume ratio of the test solution to 30-80 vol% methanol is 1:(100-500); The mass-to-volume ratio of the test solution to 50 vol% methanol is 1:(100~500); The extraction time shall not be less than 30 minutes.

4. The determination method according to claim 1, characterized in that, The reference solution was obtained by extracting a mixture of verbascoside and 30-80 vol% methanol.

5. The determination method according to claim 1, characterized in that, The amounts of the reference solution and the test solution added are each independently selected from 8 to 12 μL.

6. The determination method according to claim 5, characterized in that, The amount of both the reference solution and the test solution added was 10 μL.

7. The determination method according to claim 1, characterized in that, The column packing material in the liquid chromatography is octadecylsilane-bonded silica gel; The theoretical plate number of the chromatographic column, calculated based on the verbascoside peak, shall not be less than 3000.

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