CN112748189B - Single-needle high performance liquid chromatography identification method - Google Patents
Single-needle high performance liquid chromatography identification method Download PDFInfo
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Abstract
The invention discloses a single-side needle high performance liquid chromatography identification method. The method uses magnoflorine as reference substance, defines characteristic peak of single-side needle high performance liquid chromatography, and can be used for identifying single-side needle and distinguishing single-side needle from double-side needle. The method has the advantages of good repeatability, good stability, high accuracy, simplicity, feasibility, rapidness and low cost, and is suitable for distinguishing single-side needles from double-side needles, and quality control and authenticity identification of single-side needles. The method has the advantages of good safety, economy, environmental protection, safety, high efficiency and good application prospect.
Description
Technical Field
The invention belongs to the technical field of medicines. More particularly, relates to a single-side needle high performance liquid chromatography identification method.
Background
The Zanthoxylum nitidum (Zanthoxylum bungeanum) is Zanthoxylum bungeanum (Zanthoxylum dissitum Hemsley) and Zanthoxylum spiniferum (Z.echinocarpum Hemsley) evergreen woody lian, and the Zanthoxylum bungeanum is mainly named as Zanthoxylum bungeanum and is recorded in Chinese monograph, hunan province Chinese medicinal material standard, guangxi characteristic Chinese medicinal herb resource selective edition and other Chinese medicinal monographs. Folk therapy is commonly used for treating lumbago, toothache, traumatic injury, hernia pain, menorrhagia, etc. The root, stem and leaf can be used as medicine, has high medicinal value, and is mainly used for treating toothache, lumbago, female menorrhagia, postpartum irregular menstruation and other symptoms. The single-side needle mainly uses the clam shell pepper as the main raw material medicine of Chinese patent medicines such as gynecological Qianjin capsules, gynecological Qianjin tablets and the like, and has wide application.
The use of Zanthoxylum nitidum as a traditional Chinese medicine with a long history in China has also been recorded in the Chinese pharmacopoeia. Radix Zanthoxyli has effects of promoting blood circulation for removing blood stasis, activating qi-flowing, relieving pain, dispelling pathogenic wind, dredging collaterals, removing toxic substance and detumescence, and is mainly used for traumatic injury, stomach ache, toothache, rheumatalgia, venomous snake bite; it can be used for external treatment of burn and scald.
The single-sided needle and the double-sided needle are extremely similar in appearance and difficult to distinguish, and the chemical components of the single-sided needle and the double-sided needle are also similar. The phenomenon of serious confusion and use of single-sided needles and double-sided needles is caused because the single-sided needles lack perfect quality control standards. Therefore, the study of the single-sided needle and the double-sided needle is more and more focused by the students. For example, patent 200710064499.2 of the present applicant, in order to better control the quality of single-sided needle decoction pieces and improve the standard of single-sided needle decoction pieces, the applicant team continuously studies the standard of single-sided needle decoction pieces, improves the control index of single-sided needle decoction pieces, and effectively ensures the quality of single-sided needle decoction pieces.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography detection method for identifying single-sided needles and double-sided needles, and provides a brand new method for distinguishing the single-sided needles from the double-sided needles and for quality control and authenticity identification of single-sided needle medicinal materials. The invention obtains a liquid chromatogram characteristic peak which can be used for identifying single-sided needles and double-sided needles and controlling the quality and the authenticity of the single-sided needles through a large number of experiments, constructs a high performance liquid chromatogram detection method for identifying the single-sided needles and the double-sided needles, has good repeatability, high accuracy, simplicity, feasibility, quickness and low cost, and is suitable for distinguishing the single-sided needles and the double-sided needles and controlling the quality and identifying the authenticity of single-sided needle medicinal materials.
The above purpose of the invention is realized by the following technical scheme:
a single-side needle high performance liquid chromatography identification method comprises the following steps:
(1) Preparing a test solution: the extraction solvent is methanol, and the extraction mode is ultrasonic treatment;
(2) Preparing a magnoflorine reference substance solution;
(3) Performing high performance liquid chromatography measurement under the following chromatographic conditions:
the chromatographic column was Waters SunAire C18 (column length 25cm, inner diameter 4.6mm, particle size 5 μm);
methanol is taken as a mobile phase B, and water (0.1% formic acid) is taken as a mobile phase C;
the flow rate is 0.8-1ml/min; the column temperature is 30 ℃; the detection wavelength is 268nm;
the gradient elution procedure was:
(4) Analysis of results
In the chromatogram, the sample with characteristic peak No. 1 appearing at tR =7.0min ± 5% was a single-sided needle, and the double-sided needle had no peak No. 1.
For better control of the accuracy of the detection result, the extraction solvent in step (1) is preferably 70% methanol.
Preferably, the time of the ultrasonic treatment in the step (1) is 45-60min.
Preferably, the ultrasonic treatment in step (1) is carried out under conditions of 100 to 300W and a frequency of 30 to 50kHz.
More preferably, the ultrasonic treatment in the step (1) is ultrasonic treatment for 45min under the conditions of 200W of power and 40kHz of frequency.
Preferably, the concentration of the test solution is 0.1-0.16g/ml, and the concentration of the magnoflorine control solution is 60-100 μ g/ml.
More preferably, the concentration of the test solution is 0.1g/ml, and the concentration of the magnoflorine control solution is 60 μ g/ml.
Preferably, the amount of the sample in step (3) is 5 to 10. Mu.l (most preferably 5. Mu.l).
Preferably, the flow rate in step (3) is 1ml/min.
Preferably, the gradient elution procedure is:
in addition, as a most preferable scheme, the single-side needle high performance liquid chromatography detection method comprises the following steps:
(1) Preparation of test solution
Taking a sample of a test medicinal material, crushing, sieving by a No. 3 sieve, precisely weighing 1g of powder, placing the powder in a conical flask with a plug, precisely adding 10ml of 70% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 200W, frequency 40 kHz) for 45min, cooling, weighing, complementing the weight loss by 70% methanol, shaking up, filtering by a 0.45 mu m or 0.22 mu m microporous filter membrane, and taking a subsequent filtrate to obtain a test solution;
(2) Preparation of control solutions
Accurately weighing a proper amount of the magnoflorine reference substance dried to constant weight, placing the magnoflorine reference substance in a 10ml volumetric flask, adding 70% methanol to fully dissolve, fixing the volume to obtain a magnoflorine reference substance stock solution with the concentration of 1mg/ml, shaking up, adding 70% methanol to dilute the magnoflorine reference substance stock solution to obtain a 60 mu g/ml mixed solution, and filtering the mixed solution through a 0.45 mu m microporous filter membrane to obtain a reference substance solution;
(3) High performance liquid chromatography assay
Precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring; chromatographic conditions are as indicated above;
(4) Analysis of results
In the chromatogram, at tR =7.0min ± 5%, there was a characteristic peak unique to the first-needle from the second-needle, which was denoted as peak No. 1.
The invention has the following beneficial effects:
the invention provides a high performance liquid chromatography detection method for identifying a single-sided needle and a double-sided needle, which defines a characteristic peak (namely a No. 1 peak at the position of tR =7.0min +/-5% in a chromatogram) of the high performance liquid chromatography of the single-sided needle and can be used for identifying the single-sided needle and distinguishing the single-sided needle from the double-sided needle. The method has the advantages of good repeatability, good stability, high accuracy, simplicity, feasibility, rapidness and low cost, is suitable for distinguishing the single-side needles from the double-side needles and controlling the quality and identifying the authenticity of the single-side needles, and has good application prospect.
Drawings
FIG. 1 is a single-side needle HPLC overlay and single sample chromatogram of 30 lots in example 1.
FIG. 2 is a chromatogram of a 15 lot of the two-panel HPLC overlay and individual samples from example 1.
FIG. 3 is a single and double needle HPLC overlay and magnoflorine control chromatogram (268 nm).
FIG. 4 is a single-side needle 3D map of the detection wavelength optimization experiment of example 2.
FIG. 5 is a spectrum of magnoflorine in the detection wavelength optimization experiment of example 2.
FIG. 6 is a chromatogram of different extraction solvents for a single needle in example 2.
FIG. 7 is a comparison of chromatograms of different extraction methods for single needle in example 2.
FIG. 8 is a comparison graph of different ultrasonic time chromatograms of a single-sided needle in example 2.
FIG. 9 is the standard graph of magnoflorine in example 3.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
The following examples used the following instruments and materials:
1. instrument for measuring the position of a moving object
Waters e2695 high performance liquid chromatography system, empower workstation, containing quaternary gradient pumps, autosampler, UV detector (Waters Corp.). AB204-S type electronic balance (Mettler-Tollido instruments Co., ltd.), KQ-5200DE type numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.), DFY-300 ultra-high speed universal pulverizer (Wenling forest machines Co., ltd.), and the like.
2. Reagent
The control magnoflorine (Woodfish Biotech Co., ltd., batch No., M-026-170612, purity ≥ 98%). Methanol (Merck, germany, pure chromatography), formic acid (pure analytical chemical institute, tianjin) and water as ultrapure water.
3. Medicinal materials
The single-face needle decoction pieces are provided by Qianjin pharmaceutical industry, inc. Zanthoxylum nitidum Zanthoxylum dissatissimum Hemsley stem and Zanthoxylum nitidum (Roxb.) DC. Root of Zanthoxylum nitidum are obtained by using Zanthoxylum nitidum decoction pieces purchased from large chain drugstores, pharmaceutical companies and medicinal material markets of Hunan university of Chinese medicine university, and identified by professor Liu Dasi and professor Gong Limin subsidiary professor of Chinese medicine identification professor as shown in Table 1.
TABLE 1 sources of single and double needle samples
Example 1 Single-needle high performance liquid chromatography identification method
1. Performing high performance liquid chromatography detection on 30 batches of single-sided needles and 15 batches of double-sided needles by using magnoflorine as a reference substance, wherein the method comprises the following steps:
(1) Preparation of test solution
Taking a sample of a test sample, crushing, sieving by a No. 3 sieve, precisely weighing 1g of powder, placing the powder in a conical flask with a plug, precisely adding 10ml of 70% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 200W, frequency 40 kHz) for 45min, cooling, weighing, complementing the weight loss by 70% methanol, shaking up, filtering by a 0.45 mu m microporous filter membrane, and taking a subsequent filtrate to obtain a test sample solution (the concentration is 0.1 g/ml).
(2) Preparation of control solutions
Accurately weighing a proper amount of the magnoflorine reference substance dried to constant weight, placing the magnoflorine reference substance in a 10ml volumetric flask, adding 70% methanol to fully dissolve, fixing the volume to obtain a magnoflorine reference substance stock solution with the concentration of 1mg/ml, shaking up, adding 70% methanol to dilute the magnoflorine reference substance stock solution to obtain a 60 mu g/ml mixed solution, and filtering the mixed solution through a 0.45 mu m microporous filter membrane to obtain a reference substance solution.
(3) High performance liquid chromatography assay
Precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Chromatographic conditions are as follows:
the column was Waters SunAire C18 (column length 25cm, inner diameter 4.6mm, particle size 5 μm), methanol was used as mobile phase B, water (0.1% formic acid) was used as mobile phase C, and gradient elution was performed as shown in Table 2; the flow rate is 1ml/min; the column temperature is 30 ℃; the detection wavelength was 268nm.
The number of theoretical plates is not less than 5000 calculated according to magnoflorine.
TABLE 2 gradient elution procedure
(4) Analysis of results
In the chromatogram, at tR =7.0min ± 5%, there was a characteristic peak unique to the first-needle from the second-needle, which was denoted as peak No. 1.
2. The result of the detection
The results of the 30 single-side needle and 15 double-side needle samples are shown in fig. 1-2, and fig. 3 is a single-side needle and double-side needle HPLC overlay and magnoflorine control chromatogram (268 nm).
FIG. 1 and FIG. 2 are the superposition of characteristic maps of 30 batches of single-side needles and the superposition of characteristic maps of 15 batches of double-side needles, wherein the single-side needles mainly have a No. 1 peak and a No. 2 peak (magnoflorine), and the double-side needles only have a No. 2 peak; by comparing the patterns of the single-side needle and the double-side needle, the peak No. 1 (tR =7.0min +/-5% in chromatogram) can be used as a characteristic component for distinguishing the single-side needle from the double-side needle.
Example 2 Single-side needle high performance liquid chromatography identification method
1. Samples were randomly drawn from 30 single-side needles and 15 double-side needles in the same example 1 using magnoflorine as a control, and subjected to high performance liquid chromatography detection by the following method:
(1) Preparation of test solution
Taking a sample of a test sample, crushing, sieving by a No. 3 sieve, precisely weighing 0.12g of powder, placing in a conical flask with a plug, precisely adding 10ml of 70% methanol, sealing the plug, weighing, ultrasonically treating (with the power of 100W and the frequency of 30 kHz) for 60min, cooling, weighing, supplementing the weight loss by 70% methanol, shaking uniformly, filtering by a 0.22 mu m microporous filter membrane, and taking a subsequent filtrate to obtain a test sample solution (with the concentration of 0.12 g/ml).
(2) Preparation of control solutions
Accurately weighing a proper amount of the magnoflorine reference substance dried to constant weight, placing the magnoflorine reference substance in a 10ml volumetric flask, adding 70% methanol to fully dissolve, fixing the volume to obtain a magnoflorine reference substance stock solution with the concentration of 1mg/ml, shaking up, adding 70% methanol to dilute the magnoflorine reference substance stock solution to obtain a mixed solution with the concentration of 80 mu g/ml, and filtering the mixed solution through a 0.45 mu m microporous filter membrane to obtain a reference substance solution.
(3) High performance liquid chromatography assay
Precisely sucking 8 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Chromatographic conditions are as follows:
the column was Waters SunAir C18 (column length 25cm, inner diameter 4.6mm, particle size 5 μm), methanol was used as mobile phase B, water (0.1% formic acid) was used as mobile phase C, and gradient elution was performed as shown in Table 3; the flow rate is 1ml/min; the column temperature is 30 ℃; the detection wavelength was 268nm.
The number of theoretical plates is not less than 5000 calculated according to magnoflorine.
TABLE 3 gradient elution procedure
(4) As a result:
in the chromatogram, at tR =7.0min ± 5%, there is a No. 1 characteristic peak unique to the first needle from the second needle.
Example 3 Single-needle high performance liquid chromatography identification method
1. Samples were randomly drawn from 30 single-side needles and 15 double-side needles in the same example 1 using magnoflorine as a control, and subjected to high performance liquid chromatography detection by the following method:
(1) Preparation of test solution
Taking a sample of a test sample, crushing, sieving by a No. 3 sieve, precisely weighing 0.16g of powder, placing in a conical flask with a plug, precisely adding 10ml of 70% methanol, sealing the plug, weighing, ultrasonically treating (with the power of 300W and the frequency of 50 kHz) for 45min, cooling, weighing, supplementing the weight loss by 70% methanol, shaking uniformly, filtering by a 0.45 mu m microporous filter membrane, and taking a subsequent filtrate to obtain a test sample solution (with the concentration of 0.16 g/ml).
(2) Preparation of control solutions
Accurately weighing a proper amount of the magnoflorine reference substance dried to constant weight, placing the magnoflorine reference substance in a 10ml volumetric flask, adding 70% methanol to fully dissolve, fixing the volume to obtain a magnoflorine reference substance stock solution with the concentration of 1mg/ml, shaking up, adding 70% methanol to dilute the magnoflorine reference substance stock solution to obtain a 100 mu g/ml mixed solution, and filtering the mixed solution through a 0.45 mu m microporous filter membrane to obtain a reference substance solution.
(3) High performance liquid chromatography assay
Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Chromatographic conditions are as follows:
the column was Waters SunAire C18 (column length 25cm, inner diameter 4.6mm, particle size 5 μm), methanol was used as mobile phase B, water (0.1% formic acid) was used as mobile phase C, and gradient elution was performed as shown in Table 4; the flow rate is 0.8ml/min; the column temperature is 30 ℃; the detection wavelength was 268nm.
The number of theoretical plates is not less than 5000 calculated according to magnoflorine.
TABLE 4 gradient elution procedure
(4) As a result:
in the chromatogram, at tR =7.0min ± 5%, there is a No. 1 characteristic peak unique to the single-sided needle from the double-sided needle.
Example 4 Single-needle HPLC method optimization
1. Selection of detection wavelength
Accurately weighing appropriate amount of magnoflorine dried to constant weight, placing into 10ml volumetric flasks respectively, adding methanol to fully dissolve, fixing volume, making into magnoflorine reference substance solutions with each concentration of 1mg/ml, shaking up, and passing through 0.45 μm microporous filter membrane to obtain each reference substance stock solution. The spectrogram analysis was performed using a PDA diode array detector.
The results are shown in FIGS. 4 and 5. The result shows that the maximum absorption wavelength of the magnoflorine is 268nm.
2. Examination of extraction Process
(1) Investigation of different extraction solvents
Taking a single-sided needle sample, crushing, sieving by a third sieve, precisely weighing 1g of single-sided needle powder and 4 parts, placing the single-sided needle powder in a conical flask with a plug, precisely adding 30%, 50%, 70% and 90% methanol 10ml respectively, sealing the plug, weighing the weight, carrying out ultrasonic treatment (power 200W, frequency 40 kHz) for 45min, cooling, weighing the weight, supplementing the weight loss by 30%, 50%, 70% and 90% methanol respectively, shaking uniformly, filtering by a microporous filter membrane (0.45 mu m), taking subsequent filtrate, precisely sucking 5 mu l of the subsequent filtrate, injecting the subsequent filtrate into a liquid chromatograph, and measuring.
The results are shown in FIG. 6. The result shows that the polarity of the alkaloid is stronger, when the proportion of the organic solvent is less, the alkaloid is not beneficial to the dissolution, therefore, the extraction with 30 percent and 50 percent methanol is poorer, and the extraction rate in 70 percent methanol is higher, so the extraction solvent is extracted by 70 percent methanol.
(2) Investigation of different extraction modes
Taking a single-side needle sample, crushing, sieving by a No. 3 sieve, precisely weighing 1g of single-side needle powder and 2 parts of the single-side needle powder, placing the single-side needle powder in a conical flask with a plug, precisely adding 10ml of 70% methanol, sealing the plug, weighing, respectively carrying out ultrasonic treatment (power 200W, frequency 40 kHz) and heating reflux extraction for 45min, cooling, weighing, complementing the loss weight with 70% methanol, shaking up, filtering by a microporous filter membrane (0.45 mu m), taking subsequent filtrate, precisely sucking 5 mu l of the subsequent filtrate, injecting into a liquid chromatograph, and measuring.
The results are shown in FIG. 7. The result shows that under the ultrasonic extraction condition, the peak area is large, and the extraction rate is high, so the ultrasonic extraction is adopted as the extraction mode.
(3) Investigation of different extraction times
Taking a single-side needle sample, crushing, sieving by a No. 3 sieve, precisely weighing 1g of single-side needle powder and 4 parts of the single-side needle powder, placing the single-side needle powder in a conical flask with a plug, precisely adding 10ml of 70% methanol, sealing the plug, weighing, performing ultrasonic treatment (power 200W and frequency 40 kHz) for 25 min, 35 min, 45min and 55min respectively, cooling, weighing, supplementing the lost weight with 70% methanol, shaking up, filtering by a microporous filter membrane (0.45 mu m), taking subsequent filtrate, precisely sucking 5 mu l of the subsequent filtrate, injecting into a liquid chromatograph, and measuring.
The results are shown in FIG. 8. The result shows that ultrasonic extraction is carried out for more than 45min, the peak area is large, and the extraction rate is high. Considering all factors, the extraction time is optimally ultrasonic for 45min.
Taken together, the chromatographic procedure was determined as in example 1.
Example 5 methodological examination
1. Precision test
Taking the same batch of (S7) single-side needle samples, measuring according to the method of the embodiment 1, and continuously injecting the samples for 6 times, wherein the RSD value of the relative peak area of the magnoflorine is 0.4%, and the RSD value of the relative retention time is 0.1%, which indicates that the precision of the instrument is good.
2. Repeatability test
6 parts of the same batch (S7) of the single-side needle sample are taken, and the content RSD value of the magnoflorine is 0.19 percent and the relative retention time RSD value is 0.1 percent according to the determination method of the embodiment 1, which shows that the repeatability of the treatment method is good.
3. Stability test
According to the method of example 1, the same batch of (S7) single-side needle samples are taken to prepare the test solution, the test solution is respectively placed for 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours for measurement, and the RSD value of the peak area of the magnoflorine is 0.8 percent, which shows that the test solution has good stability within 24 hours.
4. Drawing of standard curve
Precisely transferring 1.098mg/ml of magnoflorine stock solution and 1, 1ml of magnoflorine stock solution, precisely transferring 1ml of magnoflorine stock solution 0.6477mg/ml, diluting with methanol to obtain 100, 25, 10, 5ml, 1ml and 1ml of standard solutions with the concentrations of 0.01098, 0.04392, 0.1098, 0.2196, 1.098 and 0.6477mg/ml respectively, analyzing by high performance liquid chromatography according to the chromatographic conditions of example 1, linearly regressing the concentration (X) of the standard by the peak area (Y) to obtain the regression equation Y =1.177 × 10 7 X +20338.184, r2=1.000, is well linear with peak area in the range of 10.98-1098 μ g/ml, see fig. 9.
5. Sample application recovery test
Precisely weighing 6 parts of 0.5g of powder of a single-side needle sample (S7) with known content, respectively and precisely adding 1ml of a magnoflorine reference solution with the concentration of 0.2847mg/ml, preparing a test solution according to the method of the example 1, carrying out measurement and analysis under the chromatographic condition of the example 1, and calculating the average recovery rate and RSD of each group.
The results are shown in table 5, the average sample recovery rate of magnoflorine is 96.85%, and the RSD is 0.84%, which indicates that the method has good recovery rate.
TABLE 5 magnoflorine recovery test results (n = 6)
Claims (10)
1. The method for identifying the single-side needle by high performance liquid chromatography is characterized by comprising the following steps:
(1) Preparing a test solution: the extraction solvent is methanol solution, and the extraction mode is ultrasonic treatment;
(2) Preparing a magnoflorine reference substance solution;
(3) Performing high performance liquid chromatography measurement under the following chromatographic conditions:
the chromatographic column is Waters SunAire C18, the length of the column is 25cm, the inner diameter is 4.6mm, and the particle size is 5 mu m;
methanol is taken as a mobile phase B, and 0.1% formic acid aqueous solution is taken as a mobile phase C;
the flow rate is 0.8-1ml/min; the column temperature is 30 ℃; the detection wavelength is 268nm;
the gradient elution procedure was:
(4) Analysis of results
In the chromatogram, the test sample with a characteristic peak at tR =7.0min ± 5% was a single-face needle, and the double-face needle had no such characteristic peak.
2. The method of claim 1, wherein the extraction solvent in step (1) is 70% methanol solution.
3. The method of claim 2, wherein the sonication in step (1) is performed for a period of 45min to 60min.
4. The method according to claim 3, wherein the ultrasonic treatment in the step (1) is carried out under conditions of a power of 100 to 300W and a frequency of 30 to 50kHz.
5. The method of claim 4, wherein the concentration of the test solution is 0.1-0.16g/ml and the concentration of the magnoflorine control solution is 60-100 μ g/ml.
6. The method of claim 5, wherein the concentration of the magnoflorine control solution is 60 μ g/ml.
7. The method according to claim 5, wherein the amount of the sample in the step (3) is 5 to 10. Mu.l.
8. The method of claim 7, wherein the flow rate in step (3) is 1ml/min.
9. The method of claim 8, wherein the gradient elution procedure is:
10. a method according to any one of claims 1 to 9, characterized in that the method is as follows:
(1) Preparation of test solution
Pulverizing the sample, sieving with No. 3 sieve, preparing 0.1g/ml solution with 70% methanol, ultrasonic treating at 200W and 40kHz for 45min, cooling, supplementing the lost weight with 70% methanol, shaking, filtering with 0.45 μm or 0.22 μm microporous membrane, and collecting the filtrate to obtain sample solution;
(2) Preparation of control solutions
Dissolving magnoflorine reference substance with 70% methanol to obtain magnoflorine reference substance solution with concentration of 60 μ g/ml, and filtering with 0.45 μm microporous filter membrane to obtain reference substance solution;
(3) High performance liquid chromatography assay
Injecting 5 μ l of each of the reference solution and the sample solution into a liquid chromatograph, and measuring; chromatographic conditions are as set forth in claim 1;
(4) Analysis of results
In the chromatogram, the test sample with a characteristic peak at tR =7.0min ± 5% was a single-face needle, and the double-face needle had no such characteristic peak.
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| Profiling and Pharmacokinetic Studies of Alkaloids in Rats After Oral Administration of Zanthoxylum nitidum Decoction by UPLC-Q-TOF-MS/MS and HPLC-MS/MS;Aihua Huang 等;《Molecules》;20190207;第24卷(第585期);第1-16页 * |
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