CN110658273B - Method for rapidly detecting effective components in kava pepper medicinal material - Google Patents
Method for rapidly detecting effective components in kava pepper medicinal material Download PDFInfo
- Publication number
- CN110658273B CN110658273B CN201910925187.9A CN201910925187A CN110658273B CN 110658273 B CN110658273 B CN 110658273B CN 201910925187 A CN201910925187 A CN 201910925187A CN 110658273 B CN110658273 B CN 110658273B
- Authority
- CN
- China
- Prior art keywords
- eluent
- solution
- reference substance
- sample
- chromatographic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 240000005546 Piper methysticum Species 0.000 title claims abstract description 42
- 235000016787 Piper methysticum Nutrition 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000000463 material Substances 0.000 title claims abstract description 19
- 239000013558 reference substance Substances 0.000 claims abstract description 59
- 238000001514 detection method Methods 0.000 claims abstract description 47
- 238000012937 correction Methods 0.000 claims abstract description 37
- 239000012488 sample solution Substances 0.000 claims abstract description 34
- 239000000523 sample Substances 0.000 claims abstract description 30
- 239000003480 eluent Substances 0.000 claims description 108
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 90
- 239000005711 Benzoic acid Substances 0.000 claims description 46
- 235000010233 benzoic acid Nutrition 0.000 claims description 45
- 230000014759 maintenance of location Effects 0.000 claims description 45
- XEAQIWGXBXCYFX-GUOLPTJISA-N Kawain Chemical compound C1C(OC)=CC(=O)O[C@H]1\C=C\C1=CC=CC=C1 XEAQIWGXBXCYFX-GUOLPTJISA-N 0.000 claims description 39
- XEAQIWGXBXCYFX-UHFFFAOYSA-N dl-kavain Natural products C1C(OC)=CC(=O)OC1C=CC1=CC=CC=C1 XEAQIWGXBXCYFX-UHFFFAOYSA-N 0.000 claims description 39
- RSIWXFIBHXYNFM-NSHDSACASA-N Dihydromethysticin Chemical compound C1C(OC)=CC(=O)O[C@H]1CCC1=CC=C(OCO2)C2=C1 RSIWXFIBHXYNFM-NSHDSACASA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 30
- GTEXBOVBADJOQH-FWEMWIAWSA-N Methysticin Chemical compound C1C(OC)=CC(=O)O[C@H]1\C=C\C1=CC=C(OCO2)C2=C1 GTEXBOVBADJOQH-FWEMWIAWSA-N 0.000 claims description 28
- CGGHGWCWEAXPLK-CYBMUJFWSA-N Methysticin Natural products CC(=O)C1=CC(=O)O[C@@H](C1)C=Cc2ccc3OCOc3c2 CGGHGWCWEAXPLK-CYBMUJFWSA-N 0.000 claims description 28
- 239000011550 stock solution Substances 0.000 claims description 27
- 238000004811 liquid chromatography Methods 0.000 claims description 26
- 238000010521 absorption reaction Methods 0.000 claims description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 238000002835 absorbance Methods 0.000 claims description 18
- VOOYTQRREPYRIW-LBPRGKRZSA-N Dihydrokavain Chemical compound C1C(OC)=CC(=O)O[C@H]1CCC1=CC=CC=C1 VOOYTQRREPYRIW-LBPRGKRZSA-N 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 239000000945 filler Substances 0.000 claims description 12
- 239000004480 active ingredient Substances 0.000 claims description 11
- 238000005303 weighing Methods 0.000 claims description 11
- -1 desmethoxykavain Chemical compound 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 8
- 239000012895 dilution Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 239000012925 reference material Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 239000012088 reference solution Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 238000011111 UV-scan method Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 7
- 229940075559 piperine Drugs 0.000 description 7
- 150000002559 kavalactones Chemical group 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 5
- MXXWOMGUGJBKIW-YPCIICBESA-N piperine Chemical compound C=1C=C2OCOC2=CC=1/C=C/C=C/C(=O)N1CCCCC1 MXXWOMGUGJBKIW-YPCIICBESA-N 0.000 description 5
- WVWHRXVVAYXKDE-UHFFFAOYSA-N piperine Natural products O=C(C=CC=Cc1ccc2OCOc2c1)C3CCCCN3 WVWHRXVVAYXKDE-UHFFFAOYSA-N 0.000 description 5
- 235000019100 piperine Nutrition 0.000 description 5
- VOOYTQRREPYRIW-UHFFFAOYSA-N dihydrokawain Natural products C1C(OC)=CC(=O)OC1CCC1=CC=CC=C1 VOOYTQRREPYRIW-UHFFFAOYSA-N 0.000 description 4
- 238000010812 external standard method Methods 0.000 description 4
- 238000005220 pharmaceutical analysis Methods 0.000 description 3
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- 235000013405 beer Nutrition 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000000147 hypnotic effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 238000010971 suitability test Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 208000020401 Depressive disease Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010021118 Hypotonia Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010027304 Menopausal symptoms Diseases 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 241000722363 Piper Species 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 241000758706 Piperaceae Species 0.000 description 1
- 206010039897 Sedation Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019994 cava Nutrition 0.000 description 1
- 150000001788 chalcone derivatives Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 235000019985 fermented beverage Nutrition 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000002690 local anesthesia Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036640 muscle relaxation Effects 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 229940116257 pepper extract Drugs 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000036280 sedation Effects 0.000 description 1
- 239000000932 sedative agent Substances 0.000 description 1
- 230000001624 sedative effect Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
Images
Classifications
-
- 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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- 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
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
Landscapes
- 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)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a qualitative and quantitative detection method for effective components in kava pepper medicinal materials, which specifically comprises the steps of preparing reference substance solution, determining relative correction factors of each reference substance, preparing sample solution, performing sample determination and calculating the content of the components; the method disclosed by the invention can be used for rapidly detecting the effective components and the content in the product, uses few reference substances, and can simplify the detection process, improve the detection efficiency and reduce the detection cost.
Description
Technical Field
The invention relates to the technical field of detection of active ingredients in medicinal materials, in particular to a qualitative and quantitative detection method of active ingredients in kava pepper medicinal materials.
Background
Piper methysticum is a medicinal herb plant produced in the southern Pacific region, derived from the root and rhizome of Piper methysticum Forst of Piperaceae, belonging to perennial upright shrubs, and is commonly used for preparing a non-fermented beverage with decompressing and tranquilizing effects. Cava pepper became one of the best-selling herbs in western countries, especially germany and the united states in 1995-2005, but as its application amount increased day by day, adverse reaction problems appeared gradually, in which harm to the liver was frequently reported. Modern pharmacological studies show that the kava pepper has the effects of local anesthesia, anxiety resistance, sedative hypnosis, muscle relaxation, anti-inflammation and antibiosis, climacteric symptom relief, depression treatment, convulsion, asthma and the like, and is widely used as a food supplement in recent years. But because of its serious side effects, the dosage and quality are strictly controlled.
The main effective component of the kava pepper is kava lactones accounting for 25-70% of the total extract, the kava lactones are also the most important active substances in the kava pepper, have pharmacological effects in various aspects such as sedation, hypnosis, antibiosis, pain relief and the like, and further comprise chalcone compounds. However, the prior art does not carry out comprehensive and accurate content measurement on each effective chemical compound component in the kava; in addition, the content of 6 kavalactones in the kava pepper extract is measured by adopting an external standard method in the existing content measurement, various reference substances are needed, and the reference substances are expensive and difficult to supply, so that the experiment cost is high.
Therefore, the technical personnel in the field need to solve the problem of providing a method for rapidly detecting the active ingredients in the kava pepper medicinal material in order to solve the problem.
Disclosure of Invention
In view of the above, the invention provides a method for rapidly detecting effective components in kava pepper medicinal materials, which only measures one component by utilizing the inherent functional relationship and proportional relationship of the components, realizes the synchronous control of various components, and can further improve the accuracy of detection results by adopting an internal standard which does not exist in the kava pepper medicinal materials as a reference substance; the method can quickly detect the effective components and the content in the product, uses few reference substances, and can simplify the detection process, improve the detection efficiency and reduce the detection cost.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for rapidly detecting active ingredients in a Piper methysticum Koehne medicinal material specifically comprises the following steps:
s1 reference solution
Weighing control substances including benzoic acid, methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, desmethoxykavain, kavain A and kavain B; adding solvent into the reference to obtain reference stock solution;
s2 determining relative correction factor of each control
Adding a solvent into the reference product stock solution for dilution to obtain reference product diluent, respectively determining absorbance values of the reference product diluent, and respectively calculating the percent absorption coefficient of the reference product according to the absorbance values; respectively calculating the ratio of the percent absorption coefficient of the reference substance to obtain relative correction factors by taking benzoic acid as a reference substance;
s3 sample solution preparation
Adding a sample to be detected into a benzoic acid solution to prepare a sample solution;
s4 liquid chromatography detection
Respectively carrying out liquid chromatography detection on the sample solution and the reference product storage solution to respectively obtain a reference substance, retention time of each reference substance and retention time of a sample chromatographic peak, respectively calculating the ratio of the retention time of each reference substance chromatographic peak to the retention time of the reference substance chromatographic peak to obtain relative retention time of the reference substance, determining attribution of each component in the sample solution detection chromatographic peak according to the relative retention time of the reference substance, and simultaneously reading peak areas of each component chromatographic peak to be detected and the reference substance chromatographic peak in a sample solution detection result;
s5 calculation of component content
The contents of methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, demethoxykavain, kavain A and kavain B in the sample are calculated from the relative correction factor, the peak area of the chromatographic peak of each component to be measured, the concentration of the reference stock solution and the peak area of the chromatographic peak of the reference.
Preferably, the solvent in step S1 and step S2 is the same and includes at least one of methanol, ethanol, or water.
Preferably, the solvent is added in step S1 to prepare a control stock solution with a concentration of about 0.1-1mg/ml, respectively.
Preferably, the wavelength of absorbance value detection in step S2 is 232nm, which is the same as the wavelength of liquid chromatography detection in step S4.
Preferably, in step S2, each control dilution is added into a cuvette and placed in a violet spectrophotometer, and the absorbance value of each control dilution at 232nm is measured to be 0.3-0.8.
Preferably, the percent absorption coefficient of the reference substance in step S2 is calculated according to lambert beer' S law, and the specific calculation formula is as follows:
A＝ECL，
the A is the absorbance value of each reference stock solution, the C is the concentration of each reference stock solution, and the L is the measured liquid film thickness;
the calculation formula of the relative correction factor is:
f s/x ＝E s /E x ，
f is s/x Is a relative correction factor of each component, E s Percent absorption coefficient for reference, E x Is the percentage absorption coefficient of the component to be measured.
Preferably, step S3 specifically includes: precisely weighing a sample to be measured, adding 10-50 times of solvent, performing reflux, ultrasonic treatment or cold soaking treatment, filtering, and adding a benzoic acid reference substance solution to obtain a sample solution, wherein the concentration of the benzoic acid reference substance solution is 0.1-1 mg/ml.
Preferably, the chromatographic conditions of the liquid chromatographic test in step S4 are: the filler is a chromatographic column of C18, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 100mm, the particle size of the filler is 1.7 mu m, the elution conditions are eluent A-eluent B-eluent C, and gradient elution is carried out: 70-75% of eluent A, 10% of eluent B and 20-15% of eluent C in 0-5 min; 5-11 min, wherein the eluent A is 75-65%, the eluent B is 10%, and the eluent C is 15-25%; in 11-15 min, the eluent A is 65-45%, the eluent B is 10%, and the eluent C is 25-45%; in 15-18 min, the eluent A is 45-70%, the eluent B is 10%, and the eluent C is 45-20%; the flow rate is 0.2 ml/min; sample introduction amount: 1 mul; column temperature: 35 ℃; the detection wavelength is 232 nm; the eluent A is an acetic acid aqueous solution with the concentration of 0.1%, the eluent B is acetonitrile, and the eluent C is isopropanol.
Preferably, the calculation formula of the relative retention time in step S4 is
r＝r x /r s
In the formula r x And r s The retention time of the component to be measured and the retention time of the reference substance are respectively.
Preferably, step S4 further includes a liquid chromatography system adaptability test, wherein the detection condition of the liquid chromatography system adaptability test is the same as the chromatographic condition of the liquid chromatography test.
Preferably, the calculation formula adopted in step S5 is
The W is x Is the content of the component to be measured, C x Is the concentration of the component to be measured in the sample solution
V is the volume of the sample solution, m is the sample weighing amount of the sample, A x Is the peak area of chromatographic peak of each component to be measured, C s As concentration of the reference stock solution, said f s/x Is a relative correction factor, said A s Is the peak area of the reference substance chromatographic peak in the sample solution.
According to the technical scheme, compared with the prior art, the invention discloses a qualitative and quantitative detection method for effective components in kava pepper medicinal materials, and the method has the following beneficial effects:
(1) according to the method, the percentage absorption coefficient is obtained through calculation of the absorbance value, a reference object is selected, and a relative correction factor is obtained through calculation of the percentage absorption coefficient;
(2) by establishing relative correction factors, the content of 8 components including methysticin, dihydromethysticin, kavain, dihydrokavain, kavain, desmethoxykavain, kavain A and kavain B can be simultaneously determined according to the measurement result of a reference substance in the process of determining the content of the kava components;
(3) and the content measurement result has no significant difference from an external standard method commonly used for pharmaceutical analysis, the method is simple and convenient, the accuracy is high, and the experiment cost is greatly saved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a UV scan of a benzoic acid control solution;
FIG. 2 is a UV scan of a methysticin control solution;
FIG. 3 is a UV scan of a dihydromethysticin control solution;
FIG. 4 is a UV scan of a kavain control solution;
FIG. 5 is a UV scan of a dihydrokawain control solution;
FIG. 6 is a UV scan of a kavalactone control solution;
FIG. 7 is a UV scan of a desmethoxykavain control solution;
FIG. 8 is a UV scan of a control solution of kava-piperine A;
FIG. 9 is a UV scan of a control solution of kava-kava piperine B;
FIG. 10 is an HPLC chromatogram of a benzoic acid control solution;
FIG. 11 is an HPLC chromatogram of a reference solution of methysticin;
FIG. 12 is an HPLC chromatogram of a dihydromethysticin control solution;
FIG. 13 is an HPLC chromatogram of a kavain control solution;
FIG. 14 is an HPLC chromatogram of a dihydrokawain control solution;
FIG. 15 is an HPLC chromatogram of a reference kavalactone solution;
FIG. 16 is an HPLC chromatogram of a desmethoxykavain control solution;
FIG. 17 is an HPLC chromatogram of a control solution of kava-piperine A;
FIG. 18 is an HPLC chromatogram of a control solution of kava-kava piperine B;
FIG. 19 is an HPLC chromatogram of a sample solution of Piper methysticum Forst.
In the figure 1-benzoic acid; 2-methysticin; 3 dihydromethysticin; 4-kavain; 5-dihydrokawain; 6-kavalactone; 7-desmethoxykawain; 8-kava piperine a; 9-yellow kava piperine B
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A method for rapidly detecting active ingredients in a kava pepper medicinal material specifically comprises the following steps:
s1 reference solution
Weighing control substances respectively, adding solvent to obtain control substance stock solution, wherein the control substances include benzoic acid, methysticin, dihydromethysticin, kavain, dihydrokavain, kavain, desmethoxykavain, kavain A and kavain B.
S2 determining relative correction factor of each control
Adding the solvent into the reference stock solution for dilution to obtain a reference diluent, measuring the absorbance value of the reference diluent, and respectively calculating the absorbance values to obtain the percentage absorption coefficient of the reference; taking benzoic acid as a reference substance, and respectively calculating the ratio of the percent absorption coefficient of the reference substance to obtain relative correction factors;
the percentage absorption coefficient of the reference substance is calculated according to Lambert beer law, and the specific calculation formula is as follows:
A＝ECL，
a is the absorbance value of each control stock solution, C is the concentration of each control stock solution, and L is the measured liquid film thickness;
the formula for calculating the relative correction factor is:
f s/x ＝E s /E x ，
f s/x is a relative correction factor of each component, E s Percent absorption coefficient of reference, E x Is the percentage of the component to be measuredThe absorption coefficient.
Precisely weighing a sample to be measured, adding a benzoic acid solution, carrying out reflux, ultrasonic treatment or cold soaking treatment, and filtering to obtain a clear sample solution.
S4 liquid chromatography detection
Suitability test of S41 liquid chromatography system
The detection condition of the liquid chromatography system adaptability test is the same as the chromatographic condition of the liquid chromatography detection in the sample determination process;
s42 sample determination
Respectively carrying out liquid chromatography detection on the sample solution and the reference stock solution; obtaining the retention time of a reference substance chromatographic peak, respectively calculating the ratio of the retention time of each reference substance chromatographic peak to the retention time of the reference substance chromatographic peak to obtain the relative retention time of the reference substance, determining the attribution of each component in the chromatographic peak of the sample solution to be detected according to the relative retention time of the reference substance, and simultaneously reading the peak area of each component chromatographic peak to be detected in the detection result of the sample solution and the peak area of the reference substance chromatographic peak;
the chromatographic conditions of the liquid chromatographic detection are as follows: the filler is a chromatographic column of C18, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 100mm, the particle size of the filler is 1.7 mu m, the elution conditions are eluent A-eluent B-eluent C, and gradient elution is carried out: 70-75% of eluent A, 10% of eluent B and 20-15% of eluent C in 0-5 min; 5-11 min of 75-65% of eluent A, 10% of eluent B and 15-25% of eluent C; 65-45% of eluent A, 10% of eluent B and 25-45% of eluent C in 11-15 min; 45-70% of eluent A, 10% of eluent B and 45-20% of eluent C in 15-18 min; the flow rate is 0.2 ml/min; sample introduction amount: 1 mul; column temperature: 35 ℃; the detection wavelength is 232 nm; the eluent A is an acetic acid aqueous solution with the concentration of 0.1%, the eluent B is acetonitrile, and the eluent C is isopropanol.
Wherein the calculation formula of the relative retention time is
r＝r x /r s
In the formula r x And r s The retention time of the component to be measured and the retention time of the reference substance are respectively.
Calculation of the content of S5 component
Calculating the content of methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, demethoxykavain, kavain A and kavain B in the sample respectively according to the relative correction factor, the peak area of the chromatographic peak of each component to be detected, the concentration of the reference material stock solution and the peak area of the chromatographic peak of the reference material;
wherein the calculation formula adopted is
W x Is the content of the component to be measured, C x Is the concentration of the component to be measured in the sample solution, V is the volume of the sample solution, m is the sample weighing amount, A x Is the peak area of chromatographic peak of each component to be measured, C s Concentration of stock solution as reference, f s/x Is a relative correction factor, A s Is the peak area of the reference substance chromatographic peak in the sample solution.
In order to further optimize the technical scheme, the solvent comprises at least one of methanol, ethanol or water.
For further optimization, the wavelength of absorbance value detection in step S2 is the same as the wavelength of liquid chromatography detection in step S4.
For further optimization, the detection wavelength of the absorbance value in step S2 and the detection wavelength of the liquid chromatography in step S4 are 232 nm.
In order to further optimize the technical solution, the chromatographic conditions detected by the liquid chromatography in step S4 are: the filler is a C18 chromatographic column, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 100mm, the particle size of the filler is 1.7 mu m, the elution conditions are eluent A-eluent B-eluent C, and gradient elution is carried out: 70-75% of eluent A, 10% of eluent B and 20-15% of eluent C in 0-5 min; 5-11 min of 75-65% of eluent A, 10% of eluent B and 15-25% of eluent C; 65-45% of eluent A, 10% of eluent B and 25-45% of eluent C in 11-15 min; 45-70% of eluent A, 10% of eluent B and 45-20% of eluent C in 15-18 min; the flow rate is 0.2 ml/min; sample introduction amount: 1 mul; column temperature: 35 ℃; the detection wavelength is 232 nm; the eluent A is an acetic acid aqueous solution with the concentration of 0.1%, the eluent B is acetonitrile, and the eluent C is isopropanol.
Example 1
The embodiment of the invention discloses a method for quickly detecting active ingredients in a kava pepper medicinal material, which specifically comprises the following steps:
s1 reference solution
Respectively taking benzoic acid, methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, demethoxykavain, kava-ka.
S2 determining relative correction factor of each control
Adding methanol into the above reference stock solutions for dilution, measuring absorbance value of the diluted reference stock solution at 232nm, and performing ultraviolet full-wavelength scanning on the diluted reference stock solution, with the results shown in figures 1-9; calculating the percentage absorption coefficient of the reference substance according to the absorbance values; respectively calculating the ratio of the percent absorption coefficient of the reference substance to obtain relative correction factors by taking benzoic acid as a reference substance;
the percentage absorption coefficient of the reference substance is calculated according to Lambert-beer law, and the specific calculation formula is as follows:
A＝ECL，
a is the absorbance value of each control stock solution, C is the concentration (g/ml) of each control stock solution, L is the measured liquid film thickness, and the liquid film thickness is 1 cm;
the formula for calculating the relative correction factor is:
f s/x ＝E s /E x ，
f s/x is a relative correction factor of each component, E s Percent absorption coefficient for reference, E x The percentage absorption coefficient of the component to be measured.
The final calculated relative correction factors for methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, desmethoxykavain, kava-kava B and benzoic acid are shown in table 1 below.
Table 1 relative correction factor for each component using benzoic acid as a reference (n-6)
From the results in Table 1 above, it can be seen that the relative correction factor f for methysticin Benzoic acid/methysticin Is 1.0165 relative correction factor f for dihydromethysticin Benzoic acid/dihydromethysticin Is 1.1019 relative correction factor f for kavain Benzoic acid/kavain Is 0.8969 relative correction factor f for dihydrokawain Benzoic acid/dihydrokawain 1.7629, relative correction factor f for kavalactone Benzoic acid/kava-kava Is 1.3837 relative correction factor f for desmethoxykawain Benzoic acid/desmethoxykawain 1.4459, relative correction factor f of Piperazine A Benzoic acid/kava piperine A Is 2.4218, the relative correction factor for kava-kava B/benzoic acid/kava-kava B is 2.5204.
S3 sample solution preparation
Taking at least one of kava pepper or kava pepper-containing articles as a sample, precisely weighing 0.01g of the sample, adding 10ml of methanol into the sample, ultrasonically extracting for 30min, cooling, adding methanol to make up for the lost weight, filtering, adding 1ml of benzoic acid solution with the concentration of 1mg/ml, and shaking up to obtain a sample solution;
s4 liquid chromatography detection
Suitability test of S41 liquid chromatography system
The chromatographic conditions for performing the liquid chromatographic detection are as follows: the filler is a chromatographic column of C18, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 100mm, the particle size of the filler is 1.7 mu m, the elution conditions are eluent A-eluent B-eluent C, and gradient elution is carried out: 70-75% of eluent A, 10% of eluent B and 20-15% of eluent C in 0-5 min; 5-11 min of 75-65% of eluent A, 10% of eluent B and 15-25% of eluent C; 65-45% of eluent A, 10% of eluent B and 25-45% of eluent C in 11-15 min; 45-70% of eluent A, 10% of eluent B and 45-20% of eluent C in 15-18 min; the flow rate is 0.2 ml/min; sample introduction amount: 1 mul; column temperature: 35 ℃; the detection wavelength is 232 nm; the eluent A is an acetic acid aqueous solution with the concentration of 0.1%, the eluent B is acetonitrile, and the eluent C is isopropanol.
S42 sample determination
The obtained sample solution and 9 kinds of reference stock solutions were subjected to liquid chromatography detection in an amount of 1. mu.l each, and the results are shown in FIGS. 10 to 19, wherein the detection wavelength in the liquid chromatography detection was 232nm, and the chromatographic conditions for the liquid chromatography detection were as follows: the filler is a chromatographic column of C18, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 100mm, the particle size of the filler is 1.7 mu m, the elution conditions are eluent A-eluent B-eluent C, and gradient elution is carried out: 70-75% of eluent A, 10% of eluent B and 20-15% of eluent C in 0-5 min; 5-11 min of 75-65% of eluent A, 10% of eluent B and 15-25% of eluent C; 65-45% of eluent A, 10% of eluent B and 25-45% of eluent C in 11-15 min; 45-70% of eluent A, 10% of eluent B and 45-20% of eluent C in 15-18 min; the flow rate is 0.2 ml/min; sample introduction amount: 1 mul; column temperature: 35 ℃; the detection wavelength is 232 nm; the eluent A is an acetic acid aqueous solution with the concentration of 0.1%, the eluent B is acetonitrile, and the eluent C is isopropanol.
Taking the retention time of a reference substance benzoic acid chromatographic peak as a reference retention time, and according to a calculation formula of the relative retention time
r＝r x /r s
In the formula r x And r s The retention time of the component to be measured and the retention time of the reference substance are respectively.
And calculating the ratio of the retention time of the reference substance chromatographic peak to the retention time of the reference substance benzoic acid chromatographic peak to obtain the relative retention time of the reference substance, thereby determining attribution of chromatographic peaks of 8 components to be detected, namely methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, demethoxykavain, kavain A and kavain B in the chromatographic peaks of the sample solution, and further obtaining peak areas of the chromatographic peaks of the components to be detected, wherein the relative retention time of each component to be detected and the benzoic acid reference peak is shown in the following table 2.
Table 2 relative retention time of each component with benzoic acid as reference (n ═ 6)
As shown in Table 2 above, the ratio of the methysticin chromatographic peak to benzoic acid chromatographic peak retention time is 1.5819, the ratio of the dihydromethysticin chromatographic peak to benzoic acid chromatographic peak retention time is 1.7036, the ratio of the kavain chromatographic peak to benzoic acid chromatographic peak retention time is 2.4287, the ratio of the dihydrokavain chromatographic peak to benzoic acid chromatographic peak retention time is 2.8416, the ratio of the methysticin chromatographic peak to benzoic acid chromatographic peak retention time is 3.7014, the ratio of the demethoxykavain chromatographic peak to benzoic acid chromatographic peak retention time is 4.0791, the ratio of the kava a chromatographic peak to benzoic acid chromatographic peak retention time is 6.1980, the ratio of the kava B chromatographic peak to benzoic acid chromatographic peak retention time is 6.4171, and the ratio of the benzoic acid chromatographic peak to benzoic acid chromatographic peak retention time is 1. When the content of the above 8 components in the subsequent kava pepper medicinal material is measured, the components of each chromatographic peak can be determined according to the ratio of the retention time of the chromatographic peak.
Calculation of the content of S5 component
Calculating the content of methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, demethoxykavain, kavain A and kavain B in the sample respectively according to the relative correction factor, the peak area of the chromatographic peak of each component to be detected, the concentration of the reference material stock solution and the peak area of the chromatographic peak of the reference material;
wherein the calculation formula adopted is
W x As the content of the component to be measured, C x Is the concentration of the component to be measured in the sample solution, V is the volume of the sample solution, m is the sample weighing amount, A x Is the peak area of chromatographic peak of each component to be measured, C s Concentration of stock solution as reference, f s/x Is a relative correction factor, A s Is the peak area of the reference substance chromatographic peak in the sample solution
The content of 8 ingredients in kava pepper according to the method of example 1 is shown in table 3 below.
As can be seen from the test results in Table 3, the content of 8 components in each kava pepper herb measured by the method is not significantly different from the results obtained by the external standard method commonly used in pharmaceutical analysis, i.e., the results are close to each other, which indicates that the results obtained by the method are reliable.
According to the method for quickly qualitatively and quantitatively detecting 8 effective components in the kava extract, benzoic acid which is stable in property, cheap and easy to obtain is selected as a reference substance, and a relative correction factor is calculated according to the percentage absorption coefficient of each component and the percentage absorption coefficient of an internal standard substance benzoic acid; once the relative correction factor is established, when the content of the effective components in the subsequent kava medicinal materials is measured, only 1 benzoic acid reference substance is needed, the content of 8 components such as methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, demethoxykavain, kavain A, kavain B and the like can be simultaneously measured, and the content measurement result has no significant difference from the external standard method commonly used for pharmaceutical analysis. The method is simple to operate, low in cost, and rapid and accurate.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A method for rapidly detecting active ingredients in a Piper methysticum Koehne medicinal material is characterized by comprising the following steps: s1 reference solution
Weighing control substances including benzoic acid, methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, desmethoxykavain, kavain A and kavain B; adding solvent into the reference to obtain reference stock solution;
s2 determining relative correction factor of each control
Adding a solvent into the reference product stock solution for dilution to obtain reference product diluent, respectively determining absorbance values of the reference product diluent, and respectively calculating the percent absorption coefficient of the reference product according to the absorbance values; respectively calculating the ratio of the percent absorption coefficient of the reference substance to obtain relative correction factors by taking benzoic acid as the reference substance;
s3 sample solution preparation
Adding a sample to be detected into a benzoic acid solution to prepare a sample solution; s4 liquid chromatography detection
Respectively carrying out liquid chromatography detection on the sample solution and the reference substance storage solution to respectively obtain retention time of a reference substance, each reference substance and a sample chromatographic peak, respectively calculating the ratio of the retention time of each reference substance chromatographic peak to the retention time of the reference substance chromatographic peak to obtain relative retention time of the reference substance, determining attribution of each component in the sample solution detection chromatographic peak according to the relative retention time of the reference substance, and simultaneously reading the peak area of each component chromatographic peak to be detected and the peak area of the reference substance chromatographic peak in the sample solution detection result;
s5 calculation of component content
Calculating the content of methysticin, dihydromethysticin, kavain, dihydrokavain, methysticin, demethoxykavain, kavain A and kavain B in the sample respectively according to the relative correction factor, the peak area of the chromatographic peak of each component to be detected, the concentration of the reference material stock solution and the peak area of the chromatographic peak of the reference material;
the chromatographic conditions of the liquid chromatography detection in step S4 are: the filler is a chromatographic column of C18, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 100mm, the particle size of the filler is 1.7 mu m, the elution conditions are eluent A-eluent B-eluent C, and gradient elution is carried out: 70-75% of eluent A, 10% of eluent B and 20-15% of eluent C in 0-5 min; 5-11 min of 75-65% of eluent A, 10% of eluent B and 15-25% of eluent C; in 11-15 min, the eluent A is 65-45%, the eluent B is 10%, and the eluent C is 25-45%; 45-70% of eluent A, 10% of eluent B and 45-20% of eluent C in 15-18 min; the flow rate is 0.2 ml/min; sample introduction amount: 1 mul; column temperature: 35 ℃; the detection wavelength is 232 nm; the eluent A is an acetic acid aqueous solution with the concentration of 0.1 percent, the eluent B is acetonitrile, and the eluent C is isopropanol;
the calculation formula of the relative retention time in step S4 is
r＝r x /r s
In the formula r x And r s Respectively the retention time of the component to be detected and the retention time of the reference substance;
step S4 further includes a liquid chromatography system adaptability test, where the detection conditions of the liquid chromatography system adaptability test are the same as the chromatographic conditions of the liquid chromatography detection.
2. The method as claimed in claim 1, wherein the solvent used in steps S1 and S2 is the same, and comprises at least one of methanol, ethanol or water.
3. The method for rapidly detecting the active ingredients in the kava pepper medicinal material as claimed in claim 2, wherein the solvent is added in step S1 to prepare the reference stock solutions with the concentration of 0.1-1mg/ml respectively.
4. The method as claimed in claim 1, wherein the wavelength of absorbance value detected in step S2 is 232nm, the same as the wavelength of liquid chromatography in step S4.
5. The method for rapidly detecting the active ingredients in the kava pepper medicinal material as claimed in claim 4, wherein in step S2, each control dilution is added into a cuvette and placed in a violet spectrophotometer, and the absorbance value of each control dilution at 232nm is determined to be 0.3-0.8.
6. The method for rapidly detecting the active ingredients in the kava pepper medicinal material according to claim 1, wherein the step S3 specifically comprises: precisely weighing a sample to be measured, adding 10-50 times of solvent, performing reflux, ultrasonic treatment or cold soaking treatment, filtering, and adding a benzoic acid reference substance solution to obtain a sample solution, wherein the concentration of the benzoic acid reference substance solution is 0.1-1 mg/ml.
7. The method for rapidly detecting the active ingredients in the kava pepper medicinal material as claimed in claim 1, wherein the calculation formula adopted in the step S5 is
W is x Is the content of the component to be measured, C x Is the concentration of the component to be measured in the sample solution, V is the volume of the sample solution, m is the sample weighing amount of the sample, A x Is the peak area of the chromatographic peak of each component to be measured, C s As concentration of the reference stock solution, said f s / x Is a relative correction factor, said A s Is the peak area of the reference substance chromatographic peak in the sample solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910925187.9A CN110658273B (en) | 2019-09-27 | 2019-09-27 | Method for rapidly detecting effective components in kava pepper medicinal material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910925187.9A CN110658273B (en) | 2019-09-27 | 2019-09-27 | Method for rapidly detecting effective components in kava pepper medicinal material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110658273A CN110658273A (en) | 2020-01-07 |
| CN110658273B true CN110658273B (en) | 2022-09-06 |
Family
ID=69039460
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910925187.9A Active CN110658273B (en) | 2019-09-27 | 2019-09-27 | Method for rapidly detecting effective components in kava pepper medicinal material |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110658273B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091441A (en) * | 2013-02-22 | 2013-05-08 | 贵州远程制药有限责任公司 | Quality detecting method of alpinia zerumbet medicinal material |
| EP3068416A4 (en) * | 2013-11-11 | 2017-08-09 | Kuality Herbceutics LLC | Kava derived therapeutic compounds and methods of use thereof |
-
2019
- 2019-09-27 CN CN201910925187.9A patent/CN110658273B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110658273A (en) | 2020-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102106918A (en) | Method for simultaneously preparing volatile oil, total flavones and total alkaloids from immature bitter oranges | |
| Jaiswal et al. | A Comparative Tissue‐specific Metabolite Analysis and Determination of Protodioscin Content in Asparagus Species used in Traditional Chinese Medicine and Ayurveda by use of Laser Microdissection, UHPLC–QTOF/MS and LC–MS/MS | |
| CN101703611B (en) | Quality detection method of Chinese angelica oral liquid for benefiting blood | |
| CN102749401B (en) | Inspection method of traditional Chinese medicine composition twenty-five-ingredient lung disease preparation | |
| CN103230517B (en) | A kind of decoction made from radix adenophorea, tuber of dwarf lilyturf granule and preparation method thereof and detection method | |
| CN102274401A (en) | Traditional Chinese medicine preparation for treating gastropathy and preparation method and detection method thereof | |
| CN104614450B (en) | A kind of fingerprint atlas detection method for clear preparation of quenching one's thirst | |
| CN104316613A (en) | Method for establishing fingerprint spectrum of wind-dispelling and detoxifying capsules | |
| CN102441057A (en) | High performance liquid chromatography (HPLC) fingerprint detection method for blood-nourishing brain-refreshing grain | |
| CN110658273B (en) | Method for rapidly detecting effective components in kava pepper medicinal material | |
| CN104614475B (en) | A kind of detection method of content for clear particle of quenching one's thirst | |
| Sukwattanasinit et al. | Spectrophotometric method for quantitative determination of total anthocyanins and quality characteristics of roselle (Hibiscus sabdariffa) | |
| CN104833751B (en) | The method for building up of XIAOER GANMAO TANGJIANG Aqueous extracts HPLC standard finger-print and application thereof | |
| CN110658274A (en) | Quantitative detection method for effective components in kava pepper medicinal material | |
| Rattanaburee et al. | Phytochemical study and its quantity and quality of fresh and freeze-dried young coconut juice (Cocos nucifera L.) | |
| CN101642481A (en) | Method for establishing Tripterygium wilfordii fingerprint chromatography and standard fingerprint chromatography thereof | |
| CN100570358C (en) | Treat the detection method of asthenopic pharmaceutical preparation | |
| Van Pamel et al. | Multi-class UHPLC-MS/MS method for plant toxins and cyanotoxins in food supplements and application for Belgian market samples | |
| Gondaliya et al. | Development and validation of analytical method for simultaneous estimation of berberine hydrochloride and embelin in polyherbal formulation | |
| CN102706978B (en) | Wild chrysanthemum flower extractive and fingerprint spectrum detection method thereof | |
| CN100432670C (en) | Method for inspecting Chinese-medicinal preparation Kaiyinwan | |
| CN107449848A (en) | A kind of method of La Ruia extracts in discriminating cosmetic material | |
| CN113820411A (en) | Method for measuring contents of schizandrol A and schizandrol B in preparation for strengthening body resistance and removing blood stasis | |
| Sheikh et al. | Quantitative determination of catechin as chemical marker in pediatric polyherbal syrup by HPLC/DAD | |
| Chen et al. | Reduction of β-asarone in Acori Rhizoma by Decoction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |