CN117607312A - Construction method and application of drynaria total flavone feature map - Google Patents
Construction method and application of drynaria total flavone feature map Download PDFInfo
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- 241001116742 Drynaria Species 0.000 title claims abstract description 29
- 238000010276 construction Methods 0.000 title claims abstract description 23
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 title abstract description 12
- 229930003944 flavone Natural products 0.000 title abstract description 12
- 150000002212 flavone derivatives Chemical class 0.000 title abstract description 12
- 235000011949 flavones Nutrition 0.000 title abstract description 12
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 title abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 45
- 238000000034 method Methods 0.000 claims abstract description 38
- 230000014759 maintenance of location Effects 0.000 claims abstract description 33
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 239000012488 sample solution Substances 0.000 claims abstract description 19
- 238000001228 spectrum Methods 0.000 claims abstract description 17
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000010828 elution Methods 0.000 claims abstract description 7
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 5
- 229930003935 flavonoid Natural products 0.000 claims description 29
- 150000002215 flavonoids Chemical class 0.000 claims description 29
- 235000017173 flavonoids Nutrition 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 24
- 239000001606 7-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-5-hydroxy-2-(4-hydroxyphenyl)chroman-4-one Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 21
- 229930019673 naringin Natural products 0.000 claims description 21
- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 claims description 21
- 229940052490 naringin Drugs 0.000 claims description 21
- 239000000523 sample Substances 0.000 claims description 17
- 239000012085 test solution Substances 0.000 claims description 14
- 238000000926 separation method Methods 0.000 claims description 13
- 238000012360 testing method Methods 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 238000002137 ultrasound extraction Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 4
- 239000012088 reference solution Substances 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000003908 quality control method Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 7
- 229940079593 drug Drugs 0.000 abstract description 4
- 239000000284 extract Substances 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 description 19
- 239000013558 reference substance Substances 0.000 description 12
- 238000011835 investigation Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- MZSGWZGPESCJAN-MOBFUUNNSA-N Melitric acid A Natural products O([C@@H](C(=O)O)Cc1cc(O)c(O)cc1)C(=O)/C=C/c1cc(O)c(O/C(/C(=O)O)=C/c2cc(O)c(O)cc2)cc1 MZSGWZGPESCJAN-MOBFUUNNSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- TUJPOVKMHCLXEL-UHFFFAOYSA-N eriodictyol Natural products C1C(=O)C2=CC(O)=CC(O)=C2OC1C1=CC=C(O)C(O)=C1 TUJPOVKMHCLXEL-UHFFFAOYSA-N 0.000 description 5
- SBHXYTNGIZCORC-ZDUSSCGKSA-N eriodictyol Chemical compound C1([C@@H]2CC(=O)C3=C(O)C=C(C=C3O2)O)=CC=C(O)C(O)=C1 SBHXYTNGIZCORC-ZDUSSCGKSA-N 0.000 description 5
- 235000011797 eriodictyol Nutrition 0.000 description 5
- SBHXYTNGIZCORC-UHFFFAOYSA-N eriodyctiol Natural products O1C2=CC(O)=CC(O)=C2C(=O)CC1C1=CC=C(O)C(O)=C1 SBHXYTNGIZCORC-UHFFFAOYSA-N 0.000 description 5
- 239000000126 substance Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 2
- 125000000129 anionic group Chemical group 0.000 description 2
- 210000000692 cap cell Anatomy 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 229940126680 traditional chinese medicines Drugs 0.000 description 2
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 description 1
- 241001057440 Didion Species 0.000 description 1
- JYGLAHSAISAEAL-UHFFFAOYSA-N Diphenadione Chemical compound O=C1C2=CC=CC=C2C(=O)C1C(=O)C(C=1C=CC=CC=1)C1=CC=CC=C1 JYGLAHSAISAEAL-UHFFFAOYSA-N 0.000 description 1
- 241000431987 Neocheiropteris fortunei Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002546 full scan Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 239000003643 water by type Substances 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
-
- 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
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Quality & Reliability (AREA)
- Library & Information Science (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a construction method of a drynaria total flavone characteristic map and application thereof, belonging to the technical field of detection of crude drugs or extracts, wherein the construction method comprises the steps of (1) preparing a sample solution, wherein the sample solution comprises drynaria total flavone; (2) Performing liquid chromatography detection on the sample solution, wherein the liquid chromatography detection comprises the following chromatographic conditions: mobile phase: mobile phase A is methanol, mobile phase B is 0.1% phosphoric acid solution; the gradient elution procedure was: 0-50min, the volume percentage of the mobile phase A is increased from 20% to 60%;50-60min, wherein the volume percentage of the mobile phase A is 80%;60-70min, the volume percentage of the mobile phase A is 20%. The invention obtains the characteristic spectrum of the drynaria total flavone containing 4 characteristic peaks for the first time, can monitor the quality of the medicine more intuitively, has high reliability, and has the RSD of the relative retention time of chromatographic peaks at the same position in the spectrum within 5 percent.
Description
Technical Field
The invention relates to the technical field of detection of crude drugs or extracts, in particular to a construction method of a drynaria total flavone characteristic map and application thereof.
Background
The drynaria total flavone is an effective component extracted from the traditional Chinese medicine drynaria, has the effects of tonifying kidney, strengthening bones and relieving pain, and is mainly used for clinical treatment of osteoporosis.
Traditional Chinese medicines tend to have complex ingredients, have the characteristics of multiple ingredients and multiple targets, have complex and various action mechanisms, and are common in the industry when comprehensive researches are carried out on the chemical ingredients and the pharmacodynamic substance basis of the traditional Chinese medicines with definite efficacy.
CN112946111a discloses a method for constructing and identifying a dried rhizome drynariae and its processed product UPLC fingerprint. The construction steps comprise: preparing a reference substance solution and a test substance solution respectively, and detecting the reference substance solution and the test substance solution by adopting a high performance liquid chromatography; wherein, the step of preparing the reference substance solution comprises the following steps: dissolving a reference substance, wherein the reference substance comprises 5-hydroxymethylfurfural, protocatechuic acid, new eriodictyol and naringin; the step of preparing a test solution includes: collecting rhizoma Drynariae processed product, adding solvent, extracting, filtering, and collecting filtrate, wherein the rhizoma Drynariae processed product is rhizoma Drynariae microwave processed product. The fingerprint obtained by the invention can simultaneously contain the characteristic peaks of the rhizoma drynariae and different processed products.
The main components of the rhizoma drynariae total flavonoids are naringin (accounting for 40% of the total flavonoids of the rhizoma drynariae) and new eriodictyol (accounting for 35% of the total flavonoids of the rhizoma drynariae), the proportion of the other components is relatively low, and the difference between the total flavonoids of the rhizoma drynariae and active components in rhizoma drynariae products and processed products is large, so that the construction method of the characteristic patterns or fingerprint patterns of the traditional rhizoma drynariae products and processed products cannot be fully applied to the total flavonoids of the rhizoma drynariae, and therefore, the effective quality control of the rhizoma drynariae flavonoids cannot be carried out.
Disclosure of Invention
The invention aims to provide a construction method and application of a drynaria total flavone characteristic map.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a method for constructing a characteristic map of total flavonoids of drynaria, which comprises the following steps:
(1) Preparing a test solution, wherein the test solution comprises total flavonoids of drynaria;
(2) Carrying out liquid chromatography detection on the sample solution;
the liquid chromatography detection comprises the following chromatographic conditions:
mobile phase: mobile phase A is methanol, mobile phase B is 0.1% phosphoric acid solution;
the gradient elution procedure was:
0-50min, wherein the volume percentage of the mobile phase A is increased from 20% to 60%;50-60min, wherein the volume percentage of the mobile phase A is 80%; and 60-70min, wherein the volume percentage of the mobile phase A is 20%.
The preparation method of the test sample in the step (1) comprises the steps of adding a solvent to the test sample for extraction, and carrying out solid-liquid separation, wherein the obtained liquid is the test sample solution.
Preferably, the ratio of the test sample to the solvent is 0.1:100-1000 in g/mL.
Preferably, the extraction is ultrasonic extraction, the power of the ultrasonic extraction is 450-550W, the frequency is 35-45kHz, the temperature is 20-40 ℃ and the time is more than 30 min; most preferably, the power of the ultrasonic extraction is 500W, the frequency is 40kHz, the temperature is 30 ℃, and the time is 30min.
Preferably, the solid-liquid separation is centrifugation and/or filtration.
In some embodiments, the method of preparing the test article comprises: taking 0.1g of a test sample, adding methanol, carrying out ultrasonic treatment for 30min, cooling to room temperature, fixing the volume to 100-1000mL, and carrying out solid-liquid separation to obtain filtrate, namely a test sample solution.
Preferably, the solid-liquid separation is filtration.
The chromatographic column of the liquid chromatography is octadecylsilane chemically bonded silica chromatographic column, such as Phenomenex Luna C, CAPCELL PAK MG IIC 18, tech Mate C18, kromasil 100-5C 18 And CAPCELL PAK MG IIC 18 The particle size of the filler is 3-6 μm, and the diameter of the chromatographic column4.2-4.7mm; the column length of the chromatographic column is 220-300mm.
In some embodiments, the liquid chromatography column is Phenomenex Luna C or Tech Mate C18, the packing particle size is 5 μm, the C18 silica gel bonded phase chromatography column diameter is 4.6mm; the column length of the silica gel bonded phase chromatographic column is 250mm.
The liquid chromatography detection of the invention also comprises the following chromatographic conditions:
sample injection amount: 5-10 mu L;
flow rate: 0.8-1.2mL/min;
column temperature: 25-35 ℃;
wavelength: 256nm.
Preferably, the liquid chromatography conditions are:
sample injection amount: 10. Mu.L;
flow rate: 1mL/min;
column temperature: 25 ℃;
wavelength: 256nm.
The construction method of the invention further comprises the step of carrying out liquid chromatography detection on a reference substance solution, wherein the reference substance solution contains naringin; the naringin concentration in the reference solution is 150-250 mug/mL.
Specifically, the preparation method of the reference substance solution comprises the following steps: adding naringin into methanol, mixing, and dissolving to obtain the reference solution.
In the present invention, the spectrum obtained by the liquid chromatography detection in the step (2) contains 4 common characteristic peaks, naringin is used as a reference peak S, and the relative retention time of each characteristic peak and the S peak is calculated, wherein the relative retention time is within +/-5% of a specified value, and the specified value is:
peak 1 was 0.48, peak 2 was 0.75, peak 3 was 0.84, and S peak was 1.00.
In a second aspect, the invention provides the use of the construction method in quality control of total flavonoids of drynaria.
In a third aspect, the invention provides a characteristic spectrum or a fingerprint spectrum of total flavonoids of drynaria, which is constructed by the construction method.
Specifically described are: the room temperature is 10-30 ℃; the content in the invention is mass percent unless specified otherwise.
The beneficial effects of the invention are as follows:
(1) The invention obtains the characteristic spectrum of the total flavonoids of drynaria with 4 characteristic peaks for the first time, can monitor the quality of the medicine more intuitively, and has high reliability.
(2) The relative retention time of chromatographic peaks at the same position in the spectrum is within 5%, which indicates that the spectrum has better reproducibility and has the advantages of high accuracy and high stability.
(3) The characteristic spectrum of the invention can identify the stable proportion relation of main active ingredients in the total flavonoids of drynaria, thereby ensuring the quality and the curative effect of the product.
Drawings
FIG. 1 is a characteristic spectrum of naringin of example 1;
FIG. 2 is a characteristic spectrum of total flavonoids of rhizoma Drynariae in example 1;
FIG. 3 is a control profile wherein peak 3 is eriodictyol and peak S is naringin;
FIG. 4 is a characteristic map of total flavonoids of drynaria fortunei;
FIG. 5 is a HPLC-MS total scan, wherein 5-1 is a PDA total scan, 5-2 is a cationic chromatogram, and 5-3 is an anionic chromatogram;
FIG. 6 is a 29.83min peak molecular fragment plot;
FIG. 7 is a 24.64min peak molecular fragment plot;
FIG. 8 is a diagram of a literature report of new eriodictyol molecules;
FIG. 9 is a characteristic spectrum of different column temperatures;
FIG. 10 is a characteristic spectrum of different brands of chromatographic columns.
Detailed Description
The invention is further described below in connection with examples, which are not intended to limit the scope of the invention.
The invention is further described below in connection with examples, which are not intended to limit the scope of the invention.
Instrument, reagent and sample
Agilent1100 Agilent high performance chromatograph (Instrument No.: J001); balance (Sidoris SQP/J054d=0.01 mg); ultrasonic cleaner KQ-300/J070; balance (Sidoris BS 210S/J039d=0.1 mg).
Methanol, chromatographic purity; the other reagents were all analytically pure.
Naringin (lot number 110722-200309), provided by the chinese pharmaceutical biologicals assay; rhizoma Drynariae total flavone is provided by Beijing Qihuang pharmaceutical Co.
EXAMPLE 1 construction of the Total Flavonoids characteristic Properties of drynaria
1. Preparation of control solution
Accurately weighing naringin reference substance (content 91.7%, supplied by Chinese food and drug inspection institute, batch number: 110722-201815) 20.06mg to 100ml volumetric flask, dissolving with methanol, diluting to scale, and shaking to obtain 184.0 μg/ml.
2. Preparation of test solutions
Taking 0.1g of rhizoma drynariae total flavone (batch number: 231001), precisely weighing, placing into a 100ml measuring flask, adding a proper amount of methanol, performing ultrasonic treatment (with power of 500W, frequency of 40kHz and temperature of 30 ℃) for 30 minutes, cooling to room temperature, adding methanol to dilute to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the rhizoma drynariae total flavone.
3. Construction of characteristic atlas
(1) Chromatographic conditions
Chromatographic column: tech Mate C18X 4.6mm, 120A, methanol as mobile phase A, 0.1wt% phosphoric acid solution as mobile phase B, gradient elution was performed as specified in Table 1 below; the flow rate is 1.0ml/min; the column temperature is 25 ℃, the detection wavelength is 256nm, and the theoretical plate number is not lower than 10000 according to naringin peak calculation.
TABLE 1 gradient elution table
(2) Detection and results
Precisely sucking 10 μl of each of the control solution and the sample solution, respectively, injecting into a liquid chromatograph, measuring, and recording the chromatogram.
The results are shown in FIGS. 1-3 and Table 2.
TABLE 2 results of relative retention time of characteristic peaks in the characteristic profile of total flavonoids of drynaria rhizome in unit min
From the above, it can be seen that:
(1) The peak corresponding to the reference (naringin) peak is S peak, and the relative retention time of each characteristic peak and S peak is calculated to be within + -5% of the specified value. The relative retention time specification is: 0.48 (Peak 1), 0.75 (Peak 2), 0.84 (Peak 3), 1.00 (Peak s).
(2) The relative retention time of characteristic peaks in the characteristic spectrum of the total flavonoids of drynaria rhizome is within +/-5% of a specified value, and the total flavonoids of drynaria rhizome accords with the specified value.
(3) The integral of characteristic peaks in the characteristic spectrum of the total flavonoids of drynaria rhizome of the invention has a relative minimum peak area which is more than 10 percent of the S peak area (142.787/1015.398 = 0.1406) and meets the regulations.
(4) The invention has stable detection baseline and good separation degree of target components.
Example 2 investigation of characteristic Properties of multiple batches of rhizoma Drynariae Total Flavonoids
1. Preparation of control solution
Accurately weighing naringin reference substance (content 91.7%, supplied by Chinese food and drug inspection institute, batch number: 110722-201815) 20.06mg to 100ml volumetric flask, dissolving with methanol, diluting to scale, and shaking to obtain 184.0 μg/ml.
2. Preparation of test solutions
Taking 13 batches of rhizoma Drynariae total flavonoids (batch numbers: 120901, 120902, 120903, 120904, 120905, 120906, 120907, 120908, 120909, 120910, 111214, 111215 and 111216) respectively, precisely weighing, placing into a 100ml measuring flask, adding appropriate amount of methanol, performing ultrasonic treatment (power is 500W, frequency is 40kHz, temperature is 30 ℃) for 30 minutes, cooling to 20deg.C, adding methanol to dilute to scale, shaking, filtering, and collecting the subsequent filtrate.
3. Construction of characteristic atlas
(1) Chromatographic conditions
Chromatographic column: tech Mate C18X 4.6mm, 120A, methanol as mobile phase A, 0.1wt% phosphoric acid solution as mobile phase B, gradient elution was performed as specified in Table 1 below; the flow rate is 1.0ml/min; the column temperature is 25 ℃, the detection wavelength is 256nm, and the theoretical plate number is not lower than 10000 according to naringin peak calculation.
TABLE 1 gradient elution table
(2) Detection and results
Respectively precisely sucking 10 μl of each of the sample solution and the reference solution, injecting into a liquid chromatograph, measuring, and recording the chromatogram.
The peak retention time was measured for 13 samples and the relative retention time for each peak was calculated using naringin peak (S) as a reference peak, with the results shown in Table 3 and FIG. 4.
TABLE 3 relative retention time of the common peaks of the characteristic patterns of the total flavonoids of drynaria in different lots
Conclusion:
the total flavonoids of drynaria in different batches can stably show 4 characteristic peaks, and the relative retention time of each characteristic peak and S peak is calculated to be within +/-5% of a specified value according to the measurement results of the total flavonoids of drynaria in different batches by taking naringin as a reference peak (S peak). The relative retention time specification is: 0.48 (Peak 1), 0.75 (Peak 2), 0.84 (Peak 3), 1.00 (Peak s).
Example 3 determination of characteristic Property map and identification of characteristic peaks
According to the comparison of the common peaks of 13 batches of samples, 4 characteristic peaks are determined, and the reference substance is used for identifying that the peak 4 is naringin.
Identifying other chromatographic peaks by HPLC-MS method, as shown in FIG. 5, obtaining 2 more definite chromatographic peaks in PDA full scan (see 5-1), wherein 29.83min peak is 581 (M+H+) consistent with naringin reference substance (see FIG. 6); the 24.64min peak 597 (M+H+) is consistent with the new North America eriodictyol (Neoeriocrin) reported in the literature (see FIGS. 7 and 8); other distinct chromatographic peaks were not obtained from PDA full scans (see 5-1), cationic (see 5-2) and anionic (see 5-3), nor did ion stream fragments provide more information, so no other chromatographic peaks were identified.
Example 4 examination of test sample stability
1. Preparation of control solution
The procedure is as in example 1.
2. Preparation of test solutions
The procedure is as in example 1.
3. Construction of characteristic atlas
(1) Chromatographic conditions
The procedure is as in example 1.
(2) Detection and results
The relative retention time and the relative standard deviation of the peak area were calculated by measuring 10. Mu.L of each sample solution precisely at 0, 6, 12, 18, 21 and 24 hours after preparation, and the results are shown in Table 4 and Table 5.
Table 4 stability study on retention time
TABLE 5 investigation of peak area stability
Conclusion:
the results showed that within 24 hours, the 4 common peaks had a relative retention time precision RSD of less than 0.09% and the peak areas RSD of less than 0.9%, indicating that the test solution was substantially stable within 24 hours.
Repetition of the method of example 5
1. Preparation of control solution
The procedure is as in example 1.
2. Preparation of test solutions
The procedure is as in example 1.
3. Construction of characteristic atlas
(1) Chromatographic conditions
The procedure is as in example 1.
(2) Detection and results
The relative retention time and the relative standard deviation of the peak area were calculated by measuring 6 parts of the same total flavonoids of drynaria (lot number 120901) as in example 1. The results are shown in tables 6 and 7.
Table 6 relative retention time repeatability investigation
TABLE 7 investigation of the peak area repeatability per 1g sample
Conclusion:
the results show that the relative retention time precision RSD of 4 common peaks among 6 drynaria total flavone samples is less than 0.04%, and the peak area precision RSD is less than 1.1%, which indicates that the method has good repeatability.
Example 6 precision investigation of the method
1. Preparation of control solution
The procedure is as in example 1.
2. Preparation of test solutions
The procedure is as in example 1.
3. Construction of characteristic atlas
(1) Chromatographic conditions
The procedure is as in example 1.
(2) Detection and results
10 mu L of the same sample solution of total flavonoids of drynaria (batch No. 120901) was precisely aspirated as in example 1, and the sample was continuously sampled 6 times repeatedly, and the relative standard deviation of the relative retention time and peak area was calculated between 6 needles. The results are shown in tables 8 and 9.
Table 8 relative retention time precision investigation
TABLE 9 Peak area precision investigation
Conclusion:
the results show that the relative retention time precision RSD of 4 common peaks among 6 needles is less than 0.06 percent, and the peak area precision RSD is less than 0.5 percent, which indicates that the method has good precision.
EXAMPLE 7 investigation of different column temperatures
1. Preparation of control solution
The procedure is as in example 1.
2. Preparation of test solutions
The procedure is as in example 1.
3. Construction of characteristic atlas
(1) Chromatographic conditions
The column temperatures were set at 25 ℃, 30 ℃ and 35 ℃ respectively, and the other related methods were the same as in example 1.
(2) Detection and results
10 mu L of the sample solution is precisely sucked, the sample solution is injected into a liquid chromatograph, different column temperatures are inspected, a chromatogram is recorded, and the relative standard deviation of characteristic peaks relative to retention time is calculated, and the results are shown in Table 10 and FIG. 9.
TABLE 10 relative retention time for different column temperature conditions
Conclusion:
the results show that the separation effect of each characteristic peak is basically the same at different column temperatures, and the RSD of each characteristic peak is less than 3%.
EXAMPLE 8 investigation of different liquid chromatographs
1. Preparation of control solution
The procedure is as in example 1.
2. Preparation of test solutions
The procedure is as in example 1.
3. Construction of characteristic atlas
(1) Chromatographic conditions
The methods of the other related methods are the same as in example 1, using Shimadzu 20A type, agilent1200 type high performance liquid chromatograph and Waters 2695e type high performance liquid chromatograph, respectively.
(2) Detection and results
10 mu L of the sample solution is precisely sucked, the sample solution is injected into a liquid chromatograph, different liquid chromatographs are examined, chromatograms are recorded, and the relative standard deviation of characteristic peaks relative to retention time is calculated, and the results are shown in Table 11.
TABLE 11 relative retention times of different liquid chromatographs
Conclusion:
the separation effect of each characteristic peak under different liquid chromatographs is basically the same, and the RSD of each characteristic peak is less than 3%.
EXAMPLE 9 investigation of different chromatography columns
1. Preparation of control solution
The procedure is as in example 1.
2. Preparation of test solutions
The procedure is as in example 1.
3. Construction of characteristic atlas
(1) Chromatographic conditions
The following chromatographic columns were used respectively:
①PhenomenexLunaC 18 250mm×4.6mm5µm；
②CAPCELLPAKMG ⅡC 18 250mm×4.6mm5µm；
③TechMate C18， 4.6mm×250mm 5μm；
④ Kromasil 100-5 C 18 250mm×4.6mm5µm；
⑤CAPCELLPAKMG ⅡC 18 150mm×4.6mm5µm；
the rest of the related method is the same as in example 1.
(2) Detection and results
10 mu L of the sample solution is precisely sucked, the sample solution is injected into a liquid chromatograph, different chromatographic columns are inspected, chromatograms are recorded, and the relative standard deviation of characteristic peaks relative to retention time is calculated, and the results are shown in table 12 and fig. 10.
TABLE 12 relative retention times of different chromatographic columns
Conclusion:
the results show that: the peak-out sequence of each chromatographic column is basically consistent, the column (5) is a short column with 150mm, the relative retention time of the chromatographic peak is greatly different from that of a long column with 250mm, and the separation degree is slightly poorer.
The chromatographic columns (1), 2, 3 and 4) have better separation effect on the chromatographic peaks of 1-4 (S), but have larger average difference of separation degree and retention time of other chromatographic peaks with smaller peak areas, so only the peak with the minimum peak area larger than 10% of the peak area of 4 (S) is determined and is used as a characteristic peak for analysis, the relative deviation of the characteristic peak relative to the retention time is calculated, and the result shows that the RSD of the characteristic peaks 1-4 (S) is smaller than 4%, so that the 250mm chromatographic columns of the 4 brands can be used for measuring the characteristic spectrum.
The invention has been further described above in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Claims (9)
1. The method for constructing the characteristic spectrum of the total flavonoids of drynaria is characterized by comprising the following steps of:
(1) Preparing a test solution, wherein the test solution comprises total flavonoids of drynaria;
(2) Carrying out liquid chromatography detection on the sample solution;
the liquid chromatography detection comprises the following chromatographic conditions:
mobile phase: mobile phase A is methanol, mobile phase B is 0.1% phosphoric acid solution;
the gradient elution procedure was:
0-50min, wherein the volume percentage of the mobile phase A is increased from 20% to 60%;50-60min, wherein the volume percentage of the mobile phase A is 80%; and 60-70min, wherein the volume percentage of the mobile phase A is 20%.
2. The method according to claim 1, wherein the method for preparing the sample in step (1) comprises extracting the sample with a solvent, and performing solid-liquid separation to obtain a liquid, i.e., a sample solution.
3. The method of claim 2, wherein the ratio of sample to solvent in g/mL is 0.1:100-1000;
the extraction is ultrasonic extraction, the power of the ultrasonic extraction is 450-550W, the frequency is 35-45kHz, the temperature is 20-40 ℃ and the time is more than 30 min;
the solid-liquid separation is centrifugation and/or filtration.
4. The method for constructing a sample according to claim 3, wherein the method for preparing the sample comprises: taking 0.1g of a test sample, adding methanol, carrying out ultrasonic treatment for 30min, cooling to room temperature, fixing the volume to 100-1000mL, and carrying out solid-liquid separation to obtain filtrate, namely a test sample solution.
5. The construction method according to claim 1, wherein the chromatographic column of the liquid chromatograph is an octadecylsilane chemically bonded silica chromatographic column, the filler has a particle diameter of 3-6 μm, and the chromatographic column has a diameter of 4.2-4.7mm; the column length of the chromatographic column is 220-300mm.
6. The method of claim 1, wherein the liquid chromatography detection further comprises the following chromatographic conditions:
sample injection amount: 5-10 mu L;
flow rate: 0.8-1.2mL/min;
column temperature: 25-35 ℃;
wavelength: 256nm.
7. The method of claim 1, further comprising performing a liquid chromatography test on a control solution, the control solution comprising naringin; the naringin concentration in the reference solution is 150-250 mug/mL.
8. The construction method according to any one of claims 1 to 7, wherein the liquid chromatography detected pattern of step (2) comprises 4 common characteristic peaks,
calculating the relative retention time of each characteristic peak and the S peak by taking naringin as a reference peak S, wherein the relative retention time is within +/-5% of a specified value, and the specified value is:
peak 1 was 0.48, peak 2 was 0.75, peak 3 was 0.84, and S peak was 1.00.
9. Use of the construction method according to any one of claims 1-8 for quality control of total flavonoids of drynaria.
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