CN110954643A - Method for detecting purity of pimpinellide - Google Patents
Method for detecting purity of pimpinellide Download PDFInfo
- Publication number
- CN110954643A CN110954643A CN201911323846.8A CN201911323846A CN110954643A CN 110954643 A CN110954643 A CN 110954643A CN 201911323846 A CN201911323846 A CN 201911323846A CN 110954643 A CN110954643 A CN 110954643A
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- detecting
- purity
- pimpinellide
- follows
- 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.)
- Pending
Links
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
- G01N30/89—Inverse 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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
- G01N2030/146—Preparation by elimination of some components using membranes
Abstract
The invention discloses a method for detecting the purity of pimpinella anisum lactone, which belongs to the technical field of traditional Chinese medicine purity detection and comprises the following steps: (1) and preparing a test solution: accurately weighing 1.75-3.0 mg of anise lactone sample, placing in a 1 mL or 3mL volumetric flask, fixing the volume to a scale with 50% acetonitrile-water mixed solution, shaking up, and filtering with a 0.22 μm microporous membrane or centrifuging to obtain supernatant for later use; (2) detecting the test solution by using ultra-high performance liquid chromatography and adopting gradient elution, wherein the detection conditions are as follows: a chromatographic column: a reversed phase C18 column; column temperature of the chromatographic column: 20-45 ℃; mobile phase: the mobile phase A is methanol or acetonitrile; the mobile phase B is water; flow rate: 0.2 mL/min to 0.5 mL/min; a detector: the diode array detector detects the wavelength of 254 nm-330 nm; the sample volume of the anise lactone test solution is as follows: 0.2. mu.L-1.0. mu.L. The method has the characteristics of accuracy, sensitivity and rapidness, provides reliable guarantee for impurity analysis of the pimpinellide, and saves time and material cost.
Description
Technical Field
The invention belongs to the technical field of medicine purity detection, and particularly relates to a method for detecting the purity of pimpinellide.
Background
Anisic lactone, english name: pimpinellin, chemical name: the structural formula of the 5, 6-dimethoxy-2H-furo [2, 3-H ] chromen-2-one is shown as the following formula, the anisic lactone belongs to coumarin compounds, is light yellow solid, and is a secondary metabolite of medicinal plants such as radix zanthoxyli, radix angelicae pubescentis, radix angelicae dahuricae and the like. Modern researches show that anetholide has obvious biological activity and can relieve pain (such as Mao Ying, Wang Meng, Jiamin, and the like; chemical component research of analgesic effective parts of radix angelicae dahuricae [ J ]. Chinese pharmaceutical journal, 2005(8): 583-585), hemostasis (Sun Wenbo. Toddalia asiatica root bark hemostasis active component and action mechanism research [ D ]. Guizhou medical university, 2018), and pathogenic fungus inhibition (such as Limin, Hu Jun Hua, Heyi, and the like; separation and identification of active components of citrus pathogenic fungi inhibition by radix angelicae pubescentis extract [ J ]. fruit tree academy, 2012, 29(5): 900-904 + 963).
At present, the anisic lactone as a prodrug with great development value becomes a hot point for studying by scholars at home and abroad, so the market demand for the anisic lactone is increasing day by day. However, the modern research on the fingerprint of medicinal materials of anise lactone (Qi Wei, Wang, am, etc.. 10 coumarin compounds in bamboo leaves of picrasma [ J ] the university of Chinese pharmacy, 2014, 45(3): 297-300), the research on the chemical components of botanical (Songliannah, Lvbright, Xuzenai, etc.. the research on the chemical components of Angelica sinensis root [ J ]. Chinese medicinal materials, 2014, 37(1): 55-57), and the research on pharmacology (Sun Wen Bo. Feilong blood root bark hemostasis active components and action mechanisms [ D ]. Guizhou medical university of Guizhou, 2018) are mature, and the method for detecting the purity of the anise lactone is reported. The anise lactone is derived from plants, and due to the fact that the chemical components of the plants are complex and various, and the similarity degree of partial components is high, for example, the chemical components such as the pimpinella anisum lactone and the like with extremely similar chemical structures and polarities are likely to affect the purity of the anise lactone and the like when the anise lactone and the like are prepared along with a target component due to the fact that the separation degree of the pimpinella anisum lactone is not enough in the preparation processes such as extraction, separation, purification and the like. The purity of the anise lactone is detected in a laboratory, a self-construction method is needed, an HPLC method is mostly adopted for detection at present, but the method is large in sample consumption, long in time consumption in a searching process, and wastes manpower, material resources and financial resources because the separation degree of compounds with similar properties is not as high as that of an ultra-high performance liquid chromatography (ultra-high performance liquid chromatography). Therefore, the establishment of an accurate, sensitive and rapid method for detecting the purity of the anise lactone becomes an urgent problem to be solved, and has important value on the quality control of the anise lactone monomer substance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides an accurate, sensitive and quick method for detecting the purity of the anise lactone. The purity is detected by adopting two different elution modes of two different mobile phase systems, and the two methods are mutually verified, so that the method is insufficient in complementation, high in accuracy and reliable in detection result.
The purpose of the invention is realized by the following technical scheme:
a method for detecting the purity of pimpinellide is characterized by comprising the following steps: the method comprises the following steps:
(1) and preparing a test solution: precisely weighing 1.75-3.0 mg of the anise lactone test sample, placing the test sample in a 10 mL or 3mL volumetric flask, fixing the volume to a scale by using 50% acetonitrile-water mixed solution, shaking up, and filtering through a 0.22 mu m microporous membrane or centrifuging to obtain a supernatant for later use;
(2) detecting the test solution by using ultra-high performance liquid chromatography and adopting isocratic elution or gradient elution, wherein the detection conditions are as follows:
a chromatographic column: a reversed phase C18 column;
column temperature of the chromatographic column: 20-45 ℃;
mobile phase: the mobile phase A is methanol or acetonitrile; the mobile phase B is water;
flow rate: 0.2 mL/min to 0.5 mL/min;
a detector: the diode array detector detects the wavelength of 254 nm-330 nm;
the sample volume of the anise lactone test solution is as follows: 0.2. mu.L-1.0. mu.L.
Preferably, the chromatographic column is a reversed phase C18 column, the column length is 100 mm, the inner diameter is 2.1 mm, and the silica gel particle size is 1.8 μm.
Preferably, the volume content of the methanol in the mobile phase is 50-70%, and the volume content of the water in the mobile phase is 50-30%.
Preferably, the volume of acetonitrile in the mobile phase liquid is 42-90%, and the volume of water is 58-10%.
Preferably, the methanol and the acetonitrile are chromatographically pure water, and the water is pure water.
Preferably, the gradient elution is as follows: 0 min-2.88 min, 48-58% of mobile phase A and 52-42% of mobile phase B; 2.88-3.60 min, 58-90% mobile phase A, 42-10% mobile phase B; 3.60 min-5.00 min, 90% of mobile phase A and 10% of mobile phase B; 5.00 min-5.18 min, 90-48% of mobile phase A and 10-52% of mobile phase B; 5.18 min-7.00 min, 48% mobile phase A, 52% mobile phase B.
Preferably, the gradient elution is as follows: 0 min-2.88 min, 45-52% of mobile phase A and 55-48% of mobile phase B; 2.88 min-3.60 min, 52-90% of mobile phase A and 48-10% of mobile phase B; 3.60 min-5.00 min, 90% of mobile phase A and 10% of mobile phase B; 5.00 min-5.18 min, 90-45% of mobile phase A and 10-55% of mobile phase B; 5.18 min-7.00 min, 45% mobile phase A, 55% mobile phase B.
Preferably, the isocratic elution is as follows: 0 min-7.00 min, 70% of mobile phase A and 30% of mobile phase B.
Preferably, the isocratic elution is as follows: 0 min-7.00 min, 60% of mobile phase A and 40% of mobile phase B.
Preferably, the isocratic elution is as follows: 0 min-9.00 min, 50% of mobile phase A and 50% of mobile phase B.
The beneficial effects of this technical scheme are as follows:
the purity of the anise lactone is detected by adopting the ultra-high performance liquid chromatography for the first time, the ultra-high performance liquid chromatography analysis method is high in sensitivity, good in separation degree and rapid in test, the purity is detected by adopting different elution modes of two different mobile phase systems, the two methods are mutually verified, the complementation is insufficient, the accuracy is high, and the detection result is reliable. In conclusion, the invention provides a new detection method for controlling the purity of the anise lactone monomer substance, the method has the characteristics of accuracy, sensitivity and rapidness, provides reliable guarantee for impurity analysis of the anise lactone, and saves time and material cost.
According to the invention, through a large number of practical researches, the detection conditions for detecting the purity of the anise lactone by using the ultra-high performance liquid chromatography are further obtained, and the detection conditions such as a reversed-phase C18 column, two different mobile phase systems, two different elution modes and the like are effectively and strictly controlled, so that the detection of the purity of the anise lactone by using the ultra-high performance liquid chromatography is implemented; meanwhile, the method disclosed by the invention is adopted for detection, the quantitative limit and the detection limit are low, the detection time is short, and the instrument setting and the operation are simple.
Drawings
FIG. 1 is a 254nm chromatogram of example 1;
FIG. 2 is a 305 nm chromatogram of example 1;
FIG. 3 is a 254nm chromatogram of example 2;
FIG. 4 is a 305 nm chromatogram of example 2;
FIG. 5 is a 254nm chromatogram of example 3;
FIG. 6 is a 312 nm chromatogram of example 3;
FIG. 7 is a 254nm chromatogram of example 4;
FIG. 8 is a 305 nm chromatogram of example 4;
FIG. 9 is a 254nm chromatogram of example 5;
FIG. 10 is a 315 nm chromatogram of example 5;
FIG. 11 is a 330 nm chromatogram of example 5;
FIG. 12 is a 254nm chromatogram of example 6;
FIG. 13 is a 330 nm chromatogram of example 6;
in FIGS. 1 to 13, the abscissa represents time (min) and the ordinate represents Absorbance (AU).
Detailed Description
The technical solutions for achieving the objects of the present invention are further illustrated by the following specific examples, and it should be noted that the technical solutions claimed in the present invention include, but are not limited to, the following examples.
In the invention, the anise lactone test sample comprises a chemical reference grade anise lactone (purity is 98.5%) and an anise lactone monomer obtained by separation and purification (purity is unknown).
The method is adopted to detect the purity of the anise lactone, and the precision, the stability, the detection limit and the quantification limit of an instrument are examined.
The detection conditions were as follows:
high performance liquid chromatograph: waters ACQUITY UPLC H-Class can be used;
a chromatographic column: reversed phase C18 column (Asahi Ultimate UHPLC AQ-C18, 1.8 μm, 2.1 × 100 mm, W-Port);
the mobile phase is: methanol: water = 60: 40;
the temperature of the chromatographic column is 35 ℃;
flow rate: 0.4 mL/min;
a detector: a diode array detector with a detection wavelength of 254 nm;
the sample injection amount is as follows: 0.5 mu L;
(1) examination of instrument precision
Preparing a pimpinellic lactone test solution: precisely weighing 1.75 mg of chemical reference grade anisic lactone, placing in a 10 mL volumetric flask, fixing the volume to the scale with 50% acetonitrile-water (v/v), shaking up, and filtering with a 0.22 μm microporous membrane.
Continuously feeding the prepared anise lactone test solution for 6 times, detecting and analyzing according to the detection conditions, counting the retention time and peak area of 6 times of detection in the table 1, and calculating the average value and RSD value.
From the results in table 1, it is clear that the retention time of each common peak and the RSD value of the peak area in the obtained 6 chromatograms are less than 2.0%, indicating that the instrument precision is good and meets the detection requirements.
(2) Stability examination
Preparing a pimpinellic lactone test solution: precisely weighing 1.75 mg of chemical reference grade anisic lactone, placing in a 10 mL volumetric flask, metering to the scale with methanol, shaking up, and filtering with 0.22 μm microporous membrane. And (3) respectively standing the prepared anise lactone test solution for 1 hour, 2 hours, 4 hours, 8 hours and 24 hours, carrying out detection analysis according to the detection conditions, counting the retention time and peak area of each detection in a table 2, and calculating an average value and an RSD value.
The results in table 2 show that the retention time of each common peak and the RSD value of the peak area in the obtained 6 chromatograms are less than 2.0%, which indicates that the sample is reasonably and effectively detected within 24 hours at normal temperature, and the sample has good stability and meets the detection requirements.
(3) Detection limit and quantitative limit investigation
And (3) carrying out sample injection on the standard solution of 0.32 mu g/mL once to obtain the signal-to-noise ratio S/N =11.4 (10: 1) of the sample, diluting the standard solution by 5 times, and carrying out sample injection to obtain the signal-to-noise ratio S/N =2.2 (2: 1), namely determining that the limit of quantification of the anisic lactone is 0.32 mu g/mL, and the minimum limit of detection is about 0.06 mu g/mL.
Example 1
A method for detecting the purity of pimpinellic lactone comprises the following steps of detecting by using an ultra-high performance liquid chromatography, wherein the conditions of the ultra-high performance liquid chromatography are as follows:
chromatographic conditions are as follows:
the instrument comprises the following steps: waters ACQUITY UPLC H-Class;
a chromatographic column: reversed phase C18 column (Asahi Ultimate UHPLC AQ-C18, 1.8 μm, 2.1 × 100 mm, W-Port);
the mobile phase is prepared by mixing acetonitrile and water, and adopting chromatographic pure acetonitrile and water respectively; wherein, elution is performed for 0min to 2.88 min, and column washing and column balancing are performed on the chromatographic column after elution so as to facilitate subsequent use, in the embodiment, column washing is performed for 2.88 min to 5.00min, and column balancing elution and column washing are performed for 5.00min to 7.00 min, and the elution solvent, the washing solvent and the balancing solvent adopted during specific elution, washing and balancing are operated according to the table 3;
the temperature of the chromatographic column is 35 ℃;
the flow rate is 0.3 mL/min;
a detector: diode array detectors detecting wavelengths 254nm and 305 nm;
the sample injection amount is as follows: 0.5 mu L;
in the embodiment, a pimpinellide monomer obtained by separation and purification is adopted, and the specific configuration is as follows: precisely weighing 3.0 mg of a sample, placing the sample in a 3mL volumetric flask, adding 50% acetonitrile-water (v/v) for dissolving, fixing the volume to a scale, carrying out ultrasonic treatment, shaking up, and centrifuging to obtain a supernatant.
Operating according to this example, chromatograms are shown in fig. 1 and fig. 2, and the detection results (normalization method): 93.02% (254 nm) and 86.09% (305 nm).
Example 2
A method for detecting the purity of pimpinellic lactone comprises the following steps of detecting by using an ultra-high performance liquid chromatography, wherein the conditions of the ultra-high performance liquid chromatography are as follows:
chromatographic conditions are as follows:
the instrument comprises the following steps: waters ACQUITY UPLC H-Class;
a chromatographic column: reversed phase C18 column (Asahi Ultimate UHPLC AQ-C18, 1.8 μm, 2.1 × 100 mm, W-Port);
the mobile phase is prepared by mixing acetonitrile and water, and adopting chromatographic purification and pure water respectively; wherein, elution is performed for 0min to 2.88 min, and column washing and column balancing are performed on the chromatographic column after elution so as to facilitate subsequent use, in the embodiment, column washing is performed for 2.88 min to 5.00min, and column balancing elution and column washing are performed for 5.00min to 7.00 min, and the elution solvent, the washing solvent and the balancing solvent adopted during specific elution, washing and balancing are operated according to the table 4;
the temperature of the chromatographic column is 40 ℃;
the flow rate is 0.4 mL/min;
a detector: diode array detectors detecting wavelengths 254nm and 305 nm;
the sample injection amount is as follows: 1.0 μ L;
in the embodiment, a pimpinellide monomer obtained by separation and purification is adopted, and the specific configuration is as follows: precisely weighing 3.0 mg of a sample, placing the sample in a 3mL volumetric flask, adding 50% acetonitrile-water (v/v) for dissolving, fixing the volume to a scale, carrying out ultrasonic treatment, shaking up, and centrifuging to obtain a supernatant.
By working in this example, chromatograms are obtained as shown in FIGS. 3 and 4, and the results (normalized) are 93.44% (254 nm) and 85.62% (305 nm).
Example 3
A method for detecting the purity of pimpinellic lactone comprises the following steps of detecting by using an ultra-high performance liquid chromatography, wherein the conditions of the ultra-high performance liquid chromatography are as follows:
chromatographic conditions are as follows:
the instrument comprises the following steps: waters ACQUITY UPLC H-Class;
a chromatographic column: reversed phase C18 column (Asahi Ultimate UHPLC AQ-C18, 1.8 μm, 2.1 × 100 mm, W-Port);
the mobile phase is prepared by mixing methanol and water, and adopting chromatographic purification and pure water respectively; wherein, elution is performed for 0min to 2.88 min, and column washing and column balancing are performed on the chromatographic column after elution so as to facilitate subsequent use, in the embodiment, column washing is performed for 2.88 min to 3.60min, and column balancing elution and column washing are performed for 5.00min to 7.00 min, and the elution solvent, the washing solvent and the balancing solvent adopted during specific elution, washing and balancing are operated according to the table 5;
the temperature of the chromatographic column is 25 ℃;
the flow rate is 0.4 mL/min;
a detector: diode array detectors detecting wavelengths 254nm and 312 nm;
the sample injection amount is as follows: 1.0 μ L;
in the embodiment, a pimpinellide monomer obtained by separation and purification is adopted, and the specific configuration is as follows: precisely weighing 3.0 mg of a sample, placing the sample in a 3mL volumetric flask, adding 50% acetonitrile-water (v/v) for dissolving, fixing the volume to a scale, carrying out ultrasonic treatment, shaking up, and centrifuging to obtain a supernatant.
The results of the threo spectra obtained by the procedure of this example are shown in FIGS. 5 and 6, and the results of the test (normalization): 92.65% (254 nm) and 83.85% (312 nm).
Example 4
A method for detecting the purity of pimpinellic lactone comprises the following steps of detecting by using an ultra-high performance liquid chromatography, wherein the conditions of the ultra-high performance liquid chromatography are as follows:
a chromatographic column: reversed phase C18 column (Asahi Ultimate UHPLC AQ-C18, 1.8 μm, 2.1 × 100 mm, W-Port);
mobile phase: mixing methanol and water, and respectively adopting chromatographic pure water and pure water (the volume ratio is 70: 30); eluting for 7 min;
the temperature of the chromatographic column is 20 ℃;
the flow rate is 0.2 mL/min;
a detector: a diode array detector for detecting 254nm and 305 nm of wavelength;
the sample volume of the anise lactone test solution is as follows: 0.8. mu.L.
In the embodiment, a pimpinellide monomer obtained by separation and purification is adopted, and the specific configuration is as follows: precisely weighing 3.0 mg of a sample, placing the sample in a 3mL volumetric flask, adding 50% acetonitrile-water (v/v) for dissolving, fixing the volume to a scale, carrying out ultrasonic treatment, shaking up, and centrifuging to obtain a supernatant.
Operating according to this example, the chromatograms are shown in fig. 7 and 8, and the detection results are: 93.17% (254 nm) and 86.39% (305 nm).
Example 5
A method for detecting the purity of pimpinellic lactone comprises the following steps of detecting by using an ultra-high performance liquid chromatography, wherein the conditions of the ultra-high performance liquid chromatography are as follows:
a chromatographic column: reversed phase C18 column (Asahi Ultimate UHPLC AQ-C18, 1.8 μm, 2.1 × 100 mm, W-Port);
mobile phase: mixing methanol and water, and respectively adopting chromatographic pure water and pure water (the volume ratio is 60: 40); eluting for 7 min;
the temperature of the chromatographic column is 35 ℃;
the flow rate is 0.4 mL/min;
a detector: diode array detectors detecting wavelengths 254nm, 315 nm and 330 nm;
the sample volume of the anise lactone test solution is as follows: 1.0. mu.L.
In the embodiment, a pimpinellide monomer obtained by separation and purification is adopted, and the specific configuration is as follows: precisely weighing 3.0 mg of a sample, placing the sample in a 3mL volumetric flask, adding 50% acetonitrile-water (v/v) for dissolving, fixing the volume to a scale, carrying out ultrasonic treatment, shaking up, and centrifuging to obtain a supernatant.
The chromatograms obtained by operating according to this example are shown in fig. 9-11, and the detection results are: 93.66% (254 nm), 83.94% (315 nm), 81.51% (330 nm).
Example 6
A method for detecting the purity of pimpinellic lactone comprises the following steps of detecting by using an ultra-high performance liquid chromatography, wherein the conditions of the ultra-high performance liquid chromatography are as follows:
a chromatographic column: reversed phase C18 column (Asahi Ultimate UHPLC AQ-C18, 1.8 μm, 2.1 × 100 mm, W-Port);
mobile phase: mixing methanol and water, and respectively adopting chromatographic pure water and pure water (the volume ratio is 50: 50); eluting for 9 min;
the temperature of the chromatographic column is 45 ℃;
the flow rate is 0.5 mL/min;
a detector: a diode array detector for detecting 254nm and 330 nm wavelength;
the sample volume of the anise lactone test solution is as follows: 0.2. mu.L.
In the embodiment, a pimpinellide monomer obtained by separation and purification is adopted, and the specific configuration is as follows: precisely weighing 3.0 mg of a sample, placing the sample in a 3mL volumetric flask, adding 50% acetonitrile-water (v/v) for dissolving, fixing the volume to a scale, carrying out ultrasonic treatment, shaking up, and centrifuging to obtain a supernatant.
Operating according to this example, the chromatograms are shown in fig. 11 and 12, and the detection results are: 93.13% (254 nm) and 81.12% (330 nm).
The above-mentioned embodiments are further described in detail for the purpose of illustrating the invention, the technical solutions and the advantages, it should be understood that the above-mentioned embodiments are only exemplary of the invention, and are not intended to limit the invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the invention should be included in the protection scope of the invention.
Claims (10)
1. A method for detecting the purity of pimpinellide is characterized by comprising the following steps: the method comprises the following steps:
(1) and preparing a test solution: accurately weighing 1.75-3.0 mg of anise lactone sample, placing in a 1 mL or 3mL volumetric flask, fixing the volume to a scale with 50% acetonitrile-water mixed solution, shaking up, and filtering with a 0.22 μm microporous membrane or centrifuging to obtain supernatant for later use;
(2) detecting the test solution by using ultra-high performance liquid chromatography and adopting gradient elution, wherein the detection conditions are as follows:
a chromatographic column: a reversed phase C18 column;
column temperature of the chromatographic column: 20-45 ℃;
mobile phase: the mobile phase A is methanol or acetonitrile; the mobile phase B is water;
flow rate: 0.2 mL/min to 0.5 mL/min;
a detector: the diode array detector detects the wavelength of 254 nm-330 nm;
the sample volume of the anise lactone test solution is as follows: 0.2. mu.L-1.0. mu.L.
2. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the chromatographic column is a reversed phase C18 column, the column length is 100 mm, the inner diameter is 2.1 mm, and the particle size of silica gel is 1.8 μm.
3. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the volume content of methanol in the mobile phase is 50-70%, and the volume content of water is 50-30%.
4. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the volume of acetonitrile in the mobile phase liquid is 42% -90%, and the volume of water is 58% -10%.
5. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the methanol and the acetonitrile are chromatographically pure, and the water is pure water.
6. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the gradient elutes as follows: 0 min-2.88 min, 48-58% of mobile phase A and 52-42% of mobile phase B; 2.88-3.60 min, 58-90% mobile phase A, 42-10% mobile phase B; 3.60 min-5.00 min, 90% of mobile phase A and 10% of mobile phase B; 5.00 min-5.18 min, 90-48% of mobile phase A and 10-52% of mobile phase B; 5.18 min-7.00 min, 48% mobile phase A, 52% mobile phase B.
7. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the gradient elutes as follows: 0 min-2.88 min, 45-52% of mobile phase A and 55-48% of mobile phase B; 2.88 min-3.60 min, 52-90% of mobile phase A and 48-10% of mobile phase B; 3.60 min-5.00 min, 90% of mobile phase A and 10% of mobile phase B; 5.00 min-5.18 min, 90-45% of mobile phase A and 10-55% of mobile phase B; 5.18 min-7.00 min, 45% mobile phase A, 55% mobile phase B.
8. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the isocratic elution is as follows: 0 min-7.00 min, 70% of mobile phase A and 30% of mobile phase B.
9. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the isocratic elution is as follows: 0 min-7.00 min, 60% of mobile phase A and 40% of mobile phase B.
10. The method for detecting the purity of pimpinellide according to claim 1, wherein the method comprises the following steps: the isocratic elution is as follows: 0 min-9.00 min, 50% of mobile phase A and 50% of mobile phase B.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911323846.8A CN110954643A (en) | 2019-12-20 | 2019-12-20 | Method for detecting purity of pimpinellide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911323846.8A CN110954643A (en) | 2019-12-20 | 2019-12-20 | Method for detecting purity of pimpinellide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110954643A true CN110954643A (en) | 2020-04-03 |
Family
ID=69983106
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911323846.8A Pending CN110954643A (en) | 2019-12-20 | 2019-12-20 | Method for detecting purity of pimpinellide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110954643A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102267968A (en) * | 2011-06-07 | 2011-12-07 | 南京泽朗农业发展有限公司 | Process method for preparing osthole and isopimpinellin from Cnidium |
| CN105012455A (en) * | 2015-08-19 | 2015-11-04 | 贵阳学院 | Pain-easing hemostasis medicine and preparation method and application thereof |
| CN105384748A (en) * | 2015-10-28 | 2016-03-09 | 贵阳学院 | Method for separating and purifying pimpinella anisum coumarin from Toddalia asiatica Lam and application of pimpinella anisum coumarin |
| CN105640923A (en) * | 2016-01-04 | 2016-06-08 | 贵阳学院 | Novel hemostatic adhesive plaster and preparation method thereof |
| CN107753612A (en) * | 2017-10-27 | 2018-03-06 | 贵州医科大学 | The preparation of rich furocoumarin-containing Radix Toddaliae Asiaticae extract and hemostasis medical usage |
-
2019
- 2019-12-20 CN CN201911323846.8A patent/CN110954643A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102267968A (en) * | 2011-06-07 | 2011-12-07 | 南京泽朗农业发展有限公司 | Process method for preparing osthole and isopimpinellin from Cnidium |
| CN105012455A (en) * | 2015-08-19 | 2015-11-04 | 贵阳学院 | Pain-easing hemostasis medicine and preparation method and application thereof |
| CN105384748A (en) * | 2015-10-28 | 2016-03-09 | 贵阳学院 | Method for separating and purifying pimpinella anisum coumarin from Toddalia asiatica Lam and application of pimpinella anisum coumarin |
| CN105640923A (en) * | 2016-01-04 | 2016-06-08 | 贵阳学院 | Novel hemostatic adhesive plaster and preparation method thereof |
| CN107753612A (en) * | 2017-10-27 | 2018-03-06 | 贵州医科大学 | The preparation of rich furocoumarin-containing Radix Toddaliae Asiaticae extract and hemostasis medical usage |
Non-Patent Citations (2)
| Title |
|---|
| 姚曦等: "高效液相色谱法同时测定8种丛生竹竹叶中香豆素类成分", 《食品科学》 * |
| 梁妍等: "用高效液相色谱法测定飞龙掌血中茴芹香豆素含量", 《贵阳医学院学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Simultaneous quantification of 17 constituents from Yuanhu Zhitong tablet using rapid resolution liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry | |
| CN105424850A (en) | Quality evaluation method for dendrobe medicinal material | |
| Lee et al. | Quality control of Pulsatilla koreana based on the simultaneous determination of triterpenoidal saponins by HPLC‐ELSD and principal component analysis | |
| CN102426211A (en) | Method for quantitatively determining phenolic acid compounds in barley grains | |
| CN103235050A (en) | Quality control method of panax notoginseng saponins injection | |
| CN102998383B (en) | Method for testing content of main components in Rhodiola rosea extracts | |
| Chen et al. | Determination of ellagic acid in wine by solid-phase extraction–ultra-high performance liquid chromatography–tandem mass spectrometry | |
| CN101791366A (en) | Method for testing quality of Discorea nipponica Makino in different places and medicinal materials of same genera | |
| Chen et al. | Simultaneous and highly sensitive quantification of five bioactive components in Fructus Psoraleae and in rat plasma by HPLC with fluorescence detection | |
| Zhao et al. | Quantitative analysis of five toxic alkaloids in Aconitum pendulum using ultra-performance convergence chromatography (UPC 2) coupled with mass spectrometry | |
| CN108828098B (en) | Method for determining melatonin in cotton by high performance liquid chromatography-mass spectrometry | |
| CN104931594A (en) | Method for detecting content of 5-hydroxymethyl furfural in Chinese magnoliavine | |
| CN104634911B (en) | A kind of 4 kinds of flavonoids effective constituent detection methods of CHUANKEZHI ZHUSHEYE | |
| CN110954643A (en) | Method for detecting purity of pimpinellide | |
| CN110441413A (en) | The construction method and detection method of qianbai biyan tablets HPLC finger-print | |
| Kang et al. | Simultaneous determination of three Aconitum alkaloids in six herbal medicines by high-performance liquid chromatography | |
| THU et al. | Quantitative determination of compounds from Akebia quinata by high-performance liquid chromatography | |
| CN105699581A (en) | Construction method of UPLC fingerprint of sweet clover medicinal material and standard fingerprint thereof | |
| CN101549081B (en) | Method of quality control for smilax china | |
| CN104849381A (en) | High-performance liquid chromatography-charged aerosol detector law-based method for simultaneously determining seven astragaloside components | |
| CN109557233B (en) | Method for determining content of multi-index components in white paeony root extracting solution | |
| Xia et al. | Simultaneous quantification of five dibenzocyclooctadiene lignans in Schisandra chinensis by HPLC separation and fluorescence detection | |
| Chen et al. | Determination of colchicine in mouse plasma by high performance liquid-chromatographic method with UV detection and its application to pharmacokinetic studies | |
| Guan et al. | UHPLC–MS/MS Method for Quantifying Fangchinoline, Tetrandrine and Calycosin-7-O-β-D-Glucoside of Fangji Huangqi Decoction in Rat Plasma and Its Application to a Pharmacokinetic Study | |
| CN102735766A (en) | Establishment method for polygonum perfoliatum medicinal material finger print, and standard finger print thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200403 |
|
| RJ01 | Rejection of invention patent application after publication |