CN116735749A - Method for detecting ferulic acid content in blood-replenishing particles of angelica sinensis in classical prescription - Google Patents
Method for detecting ferulic acid content in blood-replenishing particles of angelica sinensis in classical prescription Download PDFInfo
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- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 title claims abstract description 63
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 title claims abstract description 63
- 229940114124 ferulic acid Drugs 0.000 title claims abstract description 63
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 235000001785 ferulic acid Nutrition 0.000 title claims abstract description 63
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000002245 particle Substances 0.000 title claims abstract description 18
- 241000382455 Angelica sinensis Species 0.000 title abstract description 21
- 239000013558 reference substance Substances 0.000 claims abstract description 28
- 238000012360 testing method Methods 0.000 claims abstract description 20
- 239000012488 sample solution Substances 0.000 claims abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 60
- 239000000243 solution Substances 0.000 claims description 33
- 238000005303 weighing Methods 0.000 claims description 21
- 238000001514 detection method Methods 0.000 claims description 19
- 239000008187 granular material Substances 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 239000012085 test solution Substances 0.000 claims description 7
- 239000012088 reference solution Substances 0.000 claims description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 5
- 210000004369 blood Anatomy 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000000227 grinding Methods 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 2
- 241000213006 Angelica dahurica Species 0.000 claims 3
- 239000000523 sample Substances 0.000 abstract description 18
- 238000002360 preparation method Methods 0.000 abstract description 11
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 5
- 239000000047 product Substances 0.000 description 8
- 238000005259 measurement Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 238000010586 diagram Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 241000125175 Angelica Species 0.000 description 4
- 235000001287 Guettarda speciosa Nutrition 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000007667 floating Methods 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- SMDOOINVMJSDPS-UHFFFAOYSA-N Astragaloside Natural products C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)=C1 SMDOOINVMJSDPS-UHFFFAOYSA-N 0.000 description 1
- 241000045403 Astragalus propinquus Species 0.000 description 1
- 206010051625 Conjunctival hyperaemia Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- QMNWISYXSJWHRY-XWJCTJPOSA-N astragaloside Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)C4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)CC3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-XWJCTJPOSA-N 0.000 description 1
- 235000006533 astragalus Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 206010036067 polydipsia Diseases 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a method for detecting the content of ferulic acid in classical Chinese angelica blood-replenishing particles, which comprises the steps of preparation of a sample solution, preparation of a reference substance solution, chromatographic conditions, system applicability test and determination method. The method for measuring the ferulic acid limit in the sample by adopting the high performance liquid chromatography is reasonable and feasible, and the established method for measuring the ferulic acid content in the angelica sinensis blood-replenishing particles is reasonable, simple and feasible and has high accuracy.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for detecting the content of ferulic acid in blood-replenishing granules of classical Chinese angelica.
Background
The Chinese angelica blood-replenishing granule is a new developed innovation medicine of a gynaecologic classical prescription by Shanxi disc dragon pharmaceutical industry group Co-Ltd, is composed of a plurality of rare and genuine traditional Chinese medicines such as astragalus membranaceus, chinese angelica, and the like, has the efficacy of replenishing qi and promoting blood production, and is mainly used for treating blood deficiency and fever syndromes, wherein the symptoms are myofever, dryness-heat, polydipsia leading to drink, conjunctival congestion and flushing, daily and non-extinguishing pulse, large and weak pulse and heavy pressing weakness. The usage amount is as follows: orally taken, three times daily, 1 bag each time; specification of: 10g per bag.
The product is subjected to non-clinical researches such as medicinal material basic research, prescription research, medicinal material quality research, process research, quality standard research, stability research and the like by relevant technicians of the company organization, and in order to effectively control the product quality and ensure the curative effect of the product, the quality standard of the medicine is formulated according to the relevant requirements of medicine registration management method, chinese pharmacopoeia and the like, and the main content comprises prescription, preparation method, property, identification, inspection, content measurement (ferulic acid, astragaloside), functions and indications, usage, specification, storage and the like.
According to reference documents and data, there are various medicinal materials, medicines and methods for measuring the content of ferulic acid in food, but no special detection method specifically aiming at the content of ferulic acid in angelica sinensis blood-replenishing particles exists, the existing method is used in the angelica sinensis blood-replenishing particles, the specificity and the accuracy are poor, the detection requirement of the product cannot be met, and in order to accurately detect the content of ferulic acid in the angelica sinensis blood-replenishing particles, the quality of the angelica sinensis blood-replenishing particles is ensured, therefore, a detection method for the content of ferulic acid in classical angelica sinensis blood-replenishing particles is provided.
Disclosure of Invention
The invention aims to overcome the existing defects, and provides a method for detecting the content of ferulic acid in blood-replenishing granules of classical Chinese angelica, which can effectively solve the problems in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions: a method for detecting ferulic acid content in classical Chinese angelica blood-replenishing granule comprises preparing test solution, preparing reference solution, testing chromatographic condition and system applicability, and determining method;
the method for detecting the ferulic acid content in the classical Chinese angelica blood-replenishing particles comprises the following steps:
step one, preparing a sample solution: grinding appropriate amount of radix Angelicae sinensis blood replenishing granule, precisely weighing about 0.5g, placing into conical flask with plug, precisely adding 75% methanol 25ml, sealing, weighing, performing ultrasonic treatment (power 400W, frequency 40 kHz) for 30 min, cooling, weighing again, supplementing the lost weight with 75% methanol, shaking, filtering, and collecting the filtrate;
step two, preparing a reference substance solution: taking a proper amount of ferulic acid reference substance, precisely weighing, placing into a brown measuring flask, and adding 75% methanol to obtain solution containing 9ug per lml;
step three, chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filler (column length is 100mm, inner diameter is 2.lmm, and particle diameter is 2.2 μm); acetonitrile-0.085% phosphoric acid solution (35:65) is used as mobile phase; the flow rate is 0.5ml per minute; the column temperature is 30 ℃; the detection wavelength was 313nm. The theoretical plate number is not lower than 8000 calculated according to the ferulic acid peak;
step four, measuring method: precisely sucking 1u1 of each of the reference solution and the sample solution, and measuring with a liquid chromatograph.
Preferably, each bag contains 2.50-5.68mg of ferulic acid (C29H 50O); the power of ultrasonic treatment is 400W, and the frequency is 40kHz; the separation degree of the ferulic acid chromatographic peak meets the requirement.
Compared with the prior art, the invention has the beneficial effects that:
the method for measuring the ferulic acid limit in the sample by adopting the high performance liquid chromatography is reasonable and feasible, and the established method for measuring the ferulic acid content in the angelica sinensis blood-replenishing particles is reasonable, simple and feasible and has high accuracy.
Drawings
FIG. 1 is a flow chart of a method for detecting the content of ferulic acid in classical Chinese angelica blood-replenishing granules;
FIG. 2 is a linear diagram of the ferulic acid control in the present invention;
FIG. 3 is a schematic diagram showing the results of the precision test of the present invention;
FIG. 4 is a schematic representation of the results of a repeatability test according to the invention;
FIG. 5 is a schematic representation of the results of a sample recovery test in accordance with the present invention;
FIG. 6 is a schematic diagram of the result of linear investigation of the ferulic acid control in the invention;
FIG. 7 is a schematic diagram of the results of a stability test of a control solution of the present invention;
FIG. 8 is a schematic diagram showing the results of a test sample stability test according to the present invention;
FIG. 9 is a schematic representation of the results of a chromatographic condition durability test in the present invention;
FIG. 10 is a schematic diagram showing a summary of the measurement of ferulic acid in 15 batches of Angelica sinensis blood-replenishing granules in the present invention.
Detailed Description
The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1. Instrument and reagent
Instrument:
siemens Fenquishf high performance liquid chromatograph (UV detector, chromaleon 7 chromatography workstation), manufactured by Siemens Feishmania technologies Co., ltd
Evolutein 201 ultraviolet-visible spectrophotometer, manufactured by sammer, usa;
chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
SQP 1mg analytical balance, manufactured by Sidoris corporation; quinmix 213-1CN;
SQP type 0.1mg analytical balance, manufactured by Sidoris corporation; SECURA224-1CN;
SQP type 0.01mg analytical balance, manufactured by Sidoris corporation; SECURA125-1CN;
an arium (r) pro ultra-pure water machine, manufactured by Sidoris corporation;
ultrasonic cleaners, manufactured by kunshan ultrasonic instruments, inc;
ferulic acid control, lot number: 110773-201915, HPLC purity 99.4%,50 mg/min, produced in China food and drug testing institute.
Acetonitrile, chromatographic purity, aston bioengineering technology (Shanghai) Co., ltd
Methanol, produced by Anhui Tiandi high purity solvent Co., ltd;
methanol, analytically pure, produced by the company of the fine chemical industry, tianjin;
the water is ultrapure water, and the rest reagents are all analytically pure.
2. Preparation of control solution
Taking a proper amount of ferulic acid reference substance, precisely weighing, adding 75% methanol to obtain solution containing about 9ug per 1 ml.
3. Determination of the absorption maximum wavelength
9.46mg of ferulic acid reference substance is precisely weighed, 75% methanol is added for dissolution and dilution to prepare a reference substance solution with the concentration of 9.40ug/ml, the reference substance solution is scanned by an ultraviolet-visible spectrophotometer within the wavelength range of 190 nm-1100 nm, and the result shows that the ferulic acid has the maximum absorption at the wavelength of 313nm, so 313nm is taken as the measuring wavelength for measuring the ferulic acid content.
4. Chromatographic condition and System applicability test
Ferulic acid was measured by high performance liquid chromatography (general rule 0512).
Chromatographic conditions and System applicability test octadecylsilane chemically bonded silica was used as filler (column length 100mm, inner diameter 2.lmm, particle size 2.2 μm); acetonitrile-0.085% phosphoric acid solution (35:65) is used as a mobile phase; the flow rate is 0.5ml per minute; the column temperature is 30 ℃; the detection wavelength was 313nm. The theoretical plate number is not lower than 8000 calculated according to ferulic acid peak.
5. Preparation of test solutions
The preparation of the sample solution comprises taking a proper amount of the sample, grinding, taking about 0.5g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 75% methanol, sealing, weighing, performing ultrasonic treatment (power 400W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 75% methanol, shaking, filtering, and taking the subsequent filtrate.
6. Specificity test
According to the technical method under the quality standard of angelica blood-replenishing particles [ preparation method ], preparing a wine-lacking angelica negative sample solution according to the same method of preparing the sample solution, injecting 10 μl of sample solution under the established chromatographic condition, and detecting no chromatographic peak of the negative sample at the corresponding position of the ferulic acid chromatographic peak, thereby indicating that the components of other medicinal materials in the prescription have no interference on the measurement of the ferulic acid in the wine angelica under the chromatographic condition and have good specificity.
7. Precision test
The instrument repeatability is prepared according to the liquid chromatography method, the reference substance solution is prepared, the reference substance solution is respectively taken and continuously injected for 5 times, detection is carried out under the chromatographic conditions, the detection is carried out by using the peak area (Ax) of ferulic acid, and the relative standard deviation RSD after 5 sample injection measurement is 0.39%, which indicates that the precision of the instrument is good, and the reference substance is shown in figure 3.
8. Repeatability:
according to the above-mentioned liquid chromatography method, preparing 6 portions of sample solution for measuring ferulic acid content of Chinese angelica blood-replenishing granules with lot number 20220802, using same measuring personnel to make detection in same laboratory according to the above-mentioned chromatographic condition, calculating the ferulic acid content result in every sample and making investigation. Results 6 samples were measured with an average of 4.44 mg/bag and a relative standard deviation RSD of 1.80% less than 2.0% specified by the standard, indicating good reproducibility of the method, see figure 4.
9. Accuracy of
The preparation of the sample solution comprises taking a proper amount of the sample, grinding, taking about 0.5g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 75% methanol, sealing, weighing, performing ultrasonic treatment (power 400W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 75% methanol, shaking, filtering, and taking the subsequent filtrate.
Preparation of a control solution: 9.46mg (purity 99.4%) of ferulic acid reference is taken, precisely weighed, and placed in a 50ml brown measuring flask as reference stock solution. Precisely measuring 0.5ml of reference stock solution, placing into a 20ml brown measuring flask, and adding 75% methanol to obtain 9.40ug solution per lml.
Sample preparation with accuracy: taking a proper amount of the product, grinding, taking about 0.5g, precisely weighing, placing 9 parts of the product in a conical flask with a stopper, and dividing the product into three different levels of low, medium and high. The low level three groups are respectively added with 0.60ml of 188.06ug/ml of reference substance solution, and then 24.40ml of 75% methanol is measured; adding 1.25ml of the reference stock solution into the medium level three groups respectively, and weighing 23.75ml of 75% methanol; adding 1.90ml of the reference stock solution, weighing 23.10ml of 75% methanol, sealing, weighing, ultrasonic treating (power 400W, frequency 40 kHz) for 30 min, cooling, weighing, supplementing the lost weight with 75% methanol, shaking, filtering, and collecting filtrate.
Detecting according to the chromatographic conditions, calculating recovery rate according to the following formula, and calculating RSD, wherein the result is shown in figure 5;
the recovery rate test shows that the recovery rate of the added ferulic acid reference substance with three different levels of high, medium and low is between 94.15 percent and 104.01 percent, the average recovery rate is 99.77, and the recovery rate is within the range of 80 to 115 percent specified by the standard, and the test result meets the specification, so that the method is accurate and reliable, and the error is within the allowable range.
10. Limit of detection and limit of quantification
Taking 9.46mg of ferulic acid reference substance, precisely weighing, placing into a 10ml brown measuring flask, adding 75% methanol to dissolve and dilute to scale, shaking, and taking as ferulic acid reference substance stock solution. Taking 0.25ml of ferulic acid reference substance stock solution, placing into a 25ml measuring flask, adding solvent to dilute to scale, and shaking to obtain reference substance solution.
Quantitative limiting solution: and diluting and sampling the reference substance solution until the peak height is about 10 times of the noise value, thus obtaining the quantitative limiting solution.
Detection limit solution: and diluting and sampling the reference substance solution until the peak height is about 3 times of the noise value, thus obtaining the detection limit solution.
And (3) measuring: precisely sucking the quantitative limit solution and the detection limit solution respectively, injecting into a liquid chromatograph, and recording a chromatogram.
Results: the detection limit concentration of the ferulic acid reference substance solution is 0.00940ug/ml (9.40 ng); the limit concentration of the ferulic acid control solution was 0.00282ug/ml (2.82 ng).
11. Linearity of
Taking ferulic acid reference substance, preparing reference substance solutions with the concentration of 150.4ug/ml, respectively diluting into reference substance solutions with the concentrations of 37.6ug/ml, 18.8ug/ml, 9.4ug/ml, 4.7ug/ml, 2.35ug/ml and 1.175ug/ml, respectively injecting 1 ul, recording the chromatograms and peak area integral values, and carrying out parallel injection three times. Drawing a standard curve by taking the sample injection quantity (mug) of ferulic acid as an abscissa (X) and the peak area integral value of ferulic acid as an ordinate (Y), and carrying out regression analysis on the measured data to obtain a regression equation of
y= 0.9355x-0.0003, and the correlation coefficient r2=0.9999. The result shows that when the sample injection amount of the ferulic acid is between 1.175ug and 37.6ug, a good linear relation is shown between the sample injection amount and the peak area integral value, and the result is shown in figure 6 (the linear investigation result of the ferulic acid reference substance) and figure 2 (the linear investigation result of the ferulic acid reference substance).
12. Range of
According to the test results of linearity, precision and accuracy of the analysis method, the concentration range of the analysis method is determined to be 1.18 ug/ml-37.60 ug/ml, and the corresponding amount range is 1.175 mug-37.60 mug.
13. Stability test
Preparing a reference substance solution according to a formulated detection method; taking 0.5g of Angelica sinensis blood replenishing granule batch No. 20220802 sample, preparing into test solution according to the test solution preparation method, taking 1 μl of each of the reference solution and the test solution, measuring at regular intervals under the above chromatographic conditions, and examining the stability of the reference solution and the test solution, wherein the measurement results are shown in figure 7 and figure 8.
14. Durability of
Under the determined chromatographic conditions, the same reference substance and sample solution are taken, the proportion of the mobile phase and the concentration of the buffer salt are respectively subjected to fine adjustment, and the content change condition of the sample under different conditions is examined, and the result is shown in figure 9.
In the durability test of the method, the sample injection amount floats up and down by 10% on the basis of the sketch; the detection wavelength is 3nm floating up and down on the basis of the planned 313 nm; the column temperature is detected by floating up and down by 2 ℃ on the basis of the planned 30 ℃; floating 10% of the flow velocity of the mobile phase on the basis of 0.5ml/min to be planned for detection; the final result has an RSD of 1.02% and meets the specification of less than 2% when tested using chromatographic columns of different models.
In summary, octadecylsilane chemically bonded silica was used as a filler (column length of 100mm, inner diameter of 2.lmm, particle diameter of 2.2 μm) in chromatographic conditions and system applicability test; acetonitrile-0.085% phosphoric acid solution (35:65) is used as a mobile phase; the flow rate is 0.5ml per minute; the column temperature is 30 ℃; the detection wavelength was 313nm. The theoretical plate number is suitably determined by chromatographic conditions of not less than 8000 as the ferulic acid peak, and has a certain durability.
15. Sample content determination
The content data measured on the samples of the product in batches according to the method are shown in the following figure 10;
the method is used for measuring the ferulic acid content in the 15 batches of angelica blood-replenishing granule samples, and the measurement result is between 2.78mg and 5.16mg per bag. According to the measurement result of the ferulic acid content in 15 batches of angelica sinensis blood-replenishing particles, the ferulic acid content is temporarily floated by 10% at the lowest value, and floated by 10% at the high value; the content of ferulic acid in each bag is 2.50-5.68mg calculated by ferulic acid (C10H 10O 4).
The result of the methodology test shows that the limit determination method for the ferulic acid in the sample by adopting the high performance liquid chromatography is reasonable and feasible.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (2)
1. A method for detecting ferulic acid content in classical Chinese angelica blood-replenishing granule comprises preparing test solution, preparing reference solution, testing chromatographic condition and system applicability, and determining method;
the method for detecting the ferulic acid content in the classical Chinese angelica blood-replenishing particles comprises the following steps:
step one, preparing a sample solution: grinding appropriate amount of radix Angelicae sinensis blood replenishing granule, precisely weighing about 0.5g, placing into conical flask with plug, precisely adding 75% methanol 25ml, sealing, weighing, performing ultrasonic treatment (power 400W, frequency 40 kHz) for 30 min, cooling, weighing again, supplementing the lost weight with 75% methanol, shaking, filtering, and collecting the filtrate;
step two, preparing a reference substance solution: taking a proper amount of ferulic acid reference substance, precisely weighing, placing into a brown measuring flask, and adding 75% methanol to obtain solution containing 9ug per lml;
step three, chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filler (column length is 100mm, inner diameter is 2.lmm, and particle diameter is 2.2 μm); acetonitrile-0.085% phosphoric acid solution (35:65) is used as mobile phase; the flow rate is 0.5ml per minute; the column temperature is 30 ℃; the detection wavelength was 313nm. The theoretical plate number is not lower than 8000 calculated according to the ferulic acid peak; step four, measuring method: precisely sucking 1u1 of each of the reference solution and the sample solution, and measuring with a liquid chromatograph.
2. The method for detecting the ferulic acid content in the classical Chinese angelica blood-replenishing granules according to claim 1, wherein the content of the ferulic acid (C29H 50O) in each bag is 2.50-5.68 mg; the power of ultrasonic treatment is 400W, and the frequency is 40kHz; the separation degree of the ferulic acid chromatographic peak meets the requirement.
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