CN107703242A - A kind of detection method of content of psoralen in radix fid simplids simae and apiolin - Google Patents
A kind of detection method of content of psoralen in radix fid simplids simae and apiolin Download PDFInfo
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- CN107703242A CN107703242A CN201710878437.9A CN201710878437A CN107703242A CN 107703242 A CN107703242 A CN 107703242A CN 201710878437 A CN201710878437 A CN 201710878437A CN 107703242 A CN107703242 A CN 107703242A
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- apiolin
- psoralen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention discloses a kind of psoralen in radix fid simplids simae and the content assaying method of apiolin, and it uses high performance liquid chromatography, and dual wavelength determines the content of psoralen and apiolin simultaneously.It is an object of the invention to provide a kind of psoralen in radix fid simplids simae and the content assaying method of apiolin.Using high performance liquid chromatography, dual wavelength is measured to its active ingredient psoralen and apiolin, enables psoralen and apiolin to obtain most accurately examining under the conditions of optimal mobile phase, maximum absorption wavelength.Method therefor is easy, accurate, reproducible, can provide scientific basis for effectively control and the quality of evaluation radix fici simplicissimae.
Description
Technical field
The invention belongs to analytical chemistry field, and in particular to a kind of that psoralen in radix fid simplids simae and apiolin are entered simultaneously
The content assaying method of row detection.
Background technology
Radix fici simplicissimae is moraceae plants ficus simplicissima Ficus hirta Vahl. dry root[1], there is strengthening the spleen and tonifying the lung, promoting the circulation of qi
The effect of dampness removing, relaxing tendons and activating collaterals, cure mainly under powerless insufficiency of the spleen edema, deficiency of food, pulmonary tuberculosis cough, night sweat, band, postpartum agalorrhea, rheumatism numbness
Bitterly, oedema, cirrhotic ascites, hepatitis, traumatic injury etc..Radix fici simplicissimae mainly containing psoralen, cupreol, β-daucosterol,
The compounds such as hexadecylic acid, stearic acid, apiolin, cyanidenon[2,3], wherein psoralen and apiolin is its main effective active
Composition[4,5]。
Modern study shows that psoralen has antibacterial, antiviral, anticoagulation, suppresses the effect such as tumour, immunological regulation[6]。
Apiolin is a kind of common flavone compound, and pharmacological action is extensive, and external and animal experiment study finds apiolin to more
Kind tumour has prevention, treatment or Chemosensitizing effect；To the tissue such as bone, prostate, liver, eye, device caused by many reasons
The lesion of official has protective action, and Central nervous system lesion also has certain prevention effect；To female and male rat
Reproductive system endocrine function have significantly affect；In addition, apiolin also has antibacterial, antiviral, antiallergy, antioxygen
Change, protect the effect such as radiation injury and regulation differentiation[7]。
In the research of radix fici simplicissimae assay, the method for gradient elution can be used simultaneously to psoralen and apiolin
Assay is carried out, but the method operation is more complicated, wastes mobile phase.
The content of the invention
It is an object of the invention to provide a kind of psoralen in radix fid simplids simae and the quality determining method of apiolin.Utilize height
Effect liquid phase chromatogram method, dual wavelength are measured to its active ingredient psoralen and apiolin, enable psoralen and apiolin
It is enough to obtain most accurately examining under the conditions of optimal mobile phase, maximum absorption wavelength.
To achieve the above object, the technical solution used in the present invention comprises the following steps：
(1) preparation of need testing solution；
(2) content of psoralen and apiolin is determined simultaneously with high performance liquid chromatography dual wavelength, the mobile phase is
The phosphoric acid of methanol -2%, its volume ratio are 60: 40.
In step (1), radix fici simplicissimae sample being weighed, adding ethanol, be ultrasonically treated, filtering takes filtrate, and water bath method is residual
Slag adds methanol to dissolve, and produces need testing solution.
In step (1), the time of supersound process is 30min, and the temperature of water bath method is 60~70 DEG C.
The wavelength that psoralen is detected described in step (2) is 245nm, and the wavelength of the detection apiolin is 338nm.
The model of high performance liquid chromatograph used in high performance liquid chromatography described in step (2) is the systems of Agilent 1100
Row.
Chromatographic column described in step (2) is C18 posts, and its model is 250mm × 4.6mm, 5 μm.
It is 1mLmin that flow velocity is detected described in step (2)-1。
Detection temperature described in step (2) is 30 DEG C.
The present invention has the advantages that：
(1) accuracy in detection height of the invention, high sensitivity, reproducible.Psoralen 0.01045~
0.1045mg·mL-1Concentration range in, be in good linear relationship with peak area.Apiolin is in 0.0113~0.113mg
mL-1Concentration range in, be in good linear relationship with peak area.Psoralen average recovery rate is that 98.63%, RSD is
1.8% (n=6), the average recovery rate of apiolin is that 100.59%, RSD is 1.6% (n=6).
(2) present invention is a kind of easy method, uses isocratic elution, it is not necessary to using gradient elution, efficiently, conveniently,
Economy, it can be used for the quality control of radix fici simplicissimae.
(3) the inventive method goes for different high performance liquid chromatographs, breaks away from most of qualitative and quantitative detection sides
Method illustrates that the method is applied widely to specific chromatographic dependence.
Brief description of the drawings
Fig. 1 is psoralen full wavelength scanner.
Fig. 2 is apiolin full wavelength scanner.
Fig. 3 is the psoralen reference substance chromatogram (245nm) of embodiment 1.
Fig. 4 is the radix fici simplicissimae chromatogram (245nm) of embodiment 1.
Fig. 5 is the apiolin reference substance chromatogram (338nm) of embodiment 1.
Fig. 6 is the radix fici simplicissimae chromatogram (338nm) of embodiment 1.
Fig. 7 is the psoralen reference substance chromatogram (245nm) of embodiment 2.
Fig. 8 is the radix fici simplicissimae chromatogram (245nm) of embodiment 2.
Fig. 9 is the apiolin reference substance chromatogram (338nm) of embodiment 2.
Figure 10 is the radix fici simplicissimae chromatogram (338nm) of embodiment 2.
Figure 11 is the radix fici simplicissimae chromatogram (245nm) of embodiment 3.
Figure 12 is the radix fici simplicissimae chromatogram (338nm) of embodiment 3.
Embodiment
Embodiment 1
Isocratic elution determines apiolin and psoralen
Experiment material
Apiolin reference substance (Chinese solid pharmaceutical preparation manufacturing technology National Engineering Research Centre, lot number：BCTG-0516)；Mend
Bone fat element reference substance (National Institute for Food and Drugs Control, lot number：110739-201416).Radix fici simplicissimae (is rather cured Guangzhou hundred
Medicine Co., Ltd, lot number：150301 places of production：Guangdong)；Methanol (chromatographically pure, lot number：1.06007.4008 Merck KGaA company),
Remaining reagent is pure to analyze.
Laboratory apparatus
High performance liquid chromatograph (the series of Agilent 1100；Agilent G1316A pumps；The ultraviolet inspections of Agilent G1322A
Survey device；Agilent G1313A automatic samplers, Agilent companies of the U.S.)；(section's instrument is helped in Shanghai to CP225D types electronic balance
Finite instrument company)；Ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., model JP-020B).
(1) selection of wavelength is determined
Take psoralen and apiolin standard items appropriate, add methanol constant volume, in 200~400nm wave-length coverages, to Psoralen
Fat element and apiolin carry out full wavelength scanner, obtain its ultraviolet absorpting spectrum (see Fig. 1, Fig. 2), as shown in Figure 1, psoralen exists
There is absorption maximum at 245nm, therefore select Detection wavelengths of the 245nm for psoralen.As shown in Figure 2, apiolin is at 338nm
There is absorption maximum, therefore select Detection wavelengths of the 338nm for apiolin.
(2) chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler；Chromatographic column be Thermo Syncronis C18 posts (250mm ×
4.6mm, 5 μm)；With the phosphoric acid of methanol -0.2% (60: 40) for mobile phase；Detection wavelength：Psoralen 245nm, apiolin
338nm, flow velocity 1mLmin-1, detection temperature is 30 DEG C.
(3) preparation of reference substance solution
Precision weighs that psoralen reference substance is appropriate, adds methanol to be prepared into 1mL μ g containing psoralen 104.5 solution；Essence
It is close to weigh that apiolin reference substance is appropriate, add methanol to be prepared into every 1mL μ g containing apiolin 113 solution.
(4) preparation of need testing solution
Radix fici simplicissimae sample about 1.4g is taken, it is accurately weighed, put in conical flask with cover, precision adds 95% ethanol 100mL, close
Plug, weighed weight, (power 100W, frequency 40kHz) 30min is ultrasonically treated, is let cool, then weighed weight, supply less loss with ethanol
Weight, filtration, precision measures filtrate 50mL, 60~70 DEG C of water bath methods, and residue adds methanol to dissolve, is transferred to 5mL volumetric flasks
In, add methanol to shake up, produce to scale.
(5) determination method
It is accurate respectively to draw reference substance solution and each 20 μ L of need testing solution, high performance liquid chromatograph is injected, is determined, i.e.,
.
Embodiment 2
Isocratic elution determines apiolin and psoralen
Experiment material
It is same as Example 1
Laboratory apparatus
High performance liquid chromatograph (the series of Agilent 1260；Agilent G1311B pumps；The ultraviolet inspections of Agilent G4212B
Survey device；Agilent G1329B automatic samplers, AgilentG1316A column ovens, Agilent companies of the U.S.)；CP225D types electricity
Sub- balance (Shanghai You Ke instrument and meters Co., Ltd)；Ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., model JP-
020B)。
(1) selection of wavelength is determined
It is same as Example 1.
(2) chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler；Chromatographic column be HC-C18column (4.6nm × 250mm, 5 μm,
Agilent Technologies, USA (250mm × 4.6mm, 5 μm)；With the phosphoric acid of methanol -0.2% (60: 40) for mobile phase；Inspection
Survey wavelength：Psoralen 245nm, apiolin 338nm, flow velocity 1mLmin-1, detection temperature is 30 DEG C.
(3) preparation of reference substance solution
It is same as Example 1.
(4) preparation of need testing solution
It is same as Example 1.
(5) determination method
It is same as Example 1.
Embodiment 3
Gradient elution determines apiolin and psoralen
Experiment material
Apiolin reference substance (separation of Traditional Chinese Medicine University Of Guangzhou's new drug research center is made, purity 99.2%), psoralea corylifolia
Plain reference substance (Nat'l Pharmaceutical ＆ Biological Products Control Institute, lot number：110739-200309), radix fici simplicissimae medicinal material is by Heyuan City Jin Yuan
Green life Co., Ltd provides.Methanol (chromatographically pure, lot number：1.06007.4008 Merck KGaA company), remaining reagent is point
Analyse pure.
Laboratory apparatus
DIONEX SUMMIT high performance liquid chromatographs (PDA-100 detectors, UVD170U detectors, STH585 insulating boxs,
P680HPLC pumps, ASI-100 automatic samplers)；Chromeleon chromatographic work stations；KQ3200DE type medical digital controlled Ultrasound Instruments
(300W, Ultrasound Instrument Co., Ltd of city of Kunshan).
(1) chromatographic condition and system suitability：YMCC18 posts (250mm × 4.6mm, 5 μm)；Mobile phase：Methanol-
0.2% phosphoric acid, gradient elution, elution curve are shown in Table 1；Flow velocity：1.0mL/min；Column temperature：30℃；Detection wavelength：Psoralen
245nm, apiolin 338nm；Sample size：10μL.
The eluent gradient elution curve (%) of table 1
(2) preparation of reference substance solution：Precision weighs psoralen reference substance 4.18mg, puts in 10mL volumetric flasks, adds first
Alcohol shakes up, as psoralen reference substance stock solution to scale；Precision weighs apiolin reference substance 3.28mg, puts 10mL capacity
In bottle, methanol is added to be shaken up, as apiolin reference substance stock solution to scale；It is accurate respectively to draw above-mentioned psoralen reference substance
Stock solution and each 1mL of apiolin reference substance stock solution, put in 10mL volumetric flasks, add methanol dilution to produce every milliliter to scale and contain
Psoralen 0.0418mg, apiolin 0.0328mg reference substance mixed solution.
(3) preparation of need testing solution：Radix fici simplicissimae fine medicinal material powder about 2.0g is taken, it is accurately weighed, put in conical flask with cover,
Precision adds ethanol 100mL, close plug, weighed weight, is ultrasonically treated 30min, lets cool, then weighed weight, and less loss is supplied with ethanol
Weight, shake up, filter, precision measures subsequent filtrate 50mL, be evaporated, and residue adds methanol to filter, and precision measures subsequent filtrate 50mL, steams
Dry, residue adds methanol to dissolve, and is transferred in 5mL measuring bottles, adds methanol to shake up, produce to scale.
(4) determination method
It is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, high performance liquid chromatograph is injected, is determined, i.e.,
.
Embodiment 1, its research step and detailed data result are as follows：
Experiment material and instrument
Experiment material
Apiolin reference substance (Chinese solid pharmaceutical preparation manufacturing technology National Engineering Research Centre, lot number：BCTG-0516)；Mend
Bone fat element reference substance (National Institute for Food and Drugs Control, lot number：110739-201416).Radix fici simplicissimae (is rather cured Guangzhou hundred
Medicine Co., Ltd, lot number：150301 places of production：Guangdong)；Methanol (chromatographically pure, lot number：1.06007.4008 Merck KGaA company),
Remaining reagent is pure to analyze.
Laboratory apparatus
High performance liquid chromatograph (the series of Agilent 1100；Agilent G1316A pumps；The ultraviolet inspections of Agilent G1322A
Survey device；Agilent G1313A automatic samplers, Agilent companies of the U.S.)；(section's instrument is helped in Shanghai to CP225D types electronic balance
Finite instrument company)；Ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., model JP-020B).
Method validation
(1) specificity is investigated
Reference substance solution, need testing solution and each 10 μ L of negative sample solution without radix fici simplicissimae are taken, injects efficient liquid
Chromatography, in negative sample solution, there is no chromatographic peak on the position corresponding with reference substance solution, show negative sample without
Interference.
(2) investigation of instrument precision
It is accurate respectively to draw the μ L of reference substance solution 20, continuously repeat sample introduction 6 times by the content assaying method under 2.2.2 items.
The RSD=2.37% of reference substance psoralen peak area is measured, the RSD=2.90% of reference substance apiolin peak area, shows instrument
Device precision is good.
(3) linear relationship is investigated
Take psoralen reference substance to be placed in right amount in volumetric flask respectively, add methanol dilution to the concentration to be respectively
0.01045mg·mL-1、0.0209mg·mL-1、0.0418mg·mL-1、0.0627mg·mL-1、0.0836mg·mL-1With
0.1045mg·mL-1Standard liquid, by the μ L of above-mentioned chromatographic condition successively sample introduction 20, using peak area as ordinate, reference substance is dense
Spend for abscissa, draw standard curve, obtain regression equation, y=27630x+46.949, correlation coefficient r=0.9999, as a result table
Bright psoralen is in 0.01045~0.1045mgmL-1Concentration range in, be in good linear relationship with peak area.
Apiolin reference substance is taken to be placed in right amount in volumetric flask respectively, it is respectively 0.0113mg to add methanol dilution to concentration
mL-1、0.0226mg·mL-1、0.0452mg·mL-1、0.0678mg·mL-1、0.0904mg·mL-1And 0.113mgmL-1's
Standard liquid, by the μ L of above-mentioned chromatographic condition successively sample introduction 20, using peak area as ordinate, reference substance concentration is abscissa, is drawn
Standard curve, obtain regression equation, y=72102x+3.5616, correlation coefficient r=0.9998, the results showed that apiolin is 0.0113
~0.113mgmL-1Concentration range in, be in good linear relationship with peak area.
(4) repeatability is investigated
The radix fici simplicissimae about 1.4g of preparation is taken, it is totally 6 parts, accurately weighed, made by " preparation of need testing solution " under 2.2.4 items
Available test sample solution is measured, and precision is drawn 20 μ L injection high performance liquid chromatographs and is measured.Psoralea corylifolia between 6 operations
The RSD of element is 2.81%, and the RSD of apiolin is 1.41%, illustrates that this method reappearance is good.
(5) degree of accuracy (average recovery) is investigated
Radix fici simplicissimae (being 0.5494mg/g containing psoralen) about 0.7g is taken, it is totally 6 parts, accurately weighed, it is accurate respectively to add
Psoralen reference substance about 0.3540mg, it is measured by " chromatographic condition " under 2.2.2 items, calculates the rate of recovery of psoralen.
The psoralen average recovery rate measured is that 98.63%, RSD is 1.8% (n=6), and the degree of accuracy of illustration method is preferable.
The psoralen degree of accuracy experimental result of table 2
Radix fici simplicissimae (being 0.1051mg/g containing apiolin) about 0.7g is taken, it is totally 6 parts, accurately weighed, it is accurate respectively to add celery
Dish element reference substance about 0.0740mg, is measured by " chromatographic condition " under 2.2.2 items, calculates the rate of recovery of apiolin.Measure celery
The average recovery rate of dish element is that 100.59%, RSD is 1.6% (n=6), and the degree of accuracy of illustration method is preferable.
The apiolin degree of accuracy experimental result of table 3
(6) stability experiment
Radix fici simplicissimae sample is taken, precision weighs 1.4079g, puts in 100mL conical flask with cover, by the side of drafting under 2.2.4 items
Legal system available test sample solution, precision are drawn 20 μ L injection high performance liquid chromatographs, determined respectively in 0,2,4,6,8,10 hour
Its content, the average value of its peak area are：Psoralen 5891.99, apiolin 490.96, RSD are：Psoralen 1.58%,
Apiolin 2.75% (n=6), shows, sample is stable in 10 hours.
(7) assay
The content of psoralen and apiolin in 3 batches of samples is determined, the results are shown in Table 3.The psoralen of 3 batches of radix fici simplicissimaes
Average content is 0.04840mg/g, and the average content of apiolin is 0.0678mg/g.
The assay result of the psoralen of table 4, apiolin
In summary, using HPLC methods, with the phosphoric acid of methanol -0.2% (60：40) be mobile phase, at the same in 245nm and
Under 338nm, carry out assay to psoralen and apiolin, the method established, efficiently, conveniently, it is economical, can be used for five
Refer to the quality control of wild peach.When carrying out assay to radix fici simplicissimae, in terms of psoralen, content is not less than 0.3872mg/g,
In terms of apiolin, content is not less than 0.0407mg/g.
Claims (8)
1. the detection method of content of a kind of psoralen in radix fid simplids simae and apiolin, it is characterised in that comprise the following steps：
(1) preparation of need testing solution；
(2) determine the content of psoralen and apiolin simultaneously with high performance liquid chromatography dual wavelength, the mobile phase be methanol-
0.2% phosphoric acid, its volume ratio are 60: 40.
2. according to the method for claim 1, it is characterised in that in the step (1), weigh radix fici simplicissimae sample, add
Ethanol, it is ultrasonically treated, filtering, takes filtrate, water bath method, residue adds methanol to dissolve, and produces need testing solution.
3. according to the method for claim 2, it is characterised in that in the step (1), the time of supersound process is 30min,
The temperature of water bath method is 60~70 DEG C.
4. according to the method for claim 1, it is characterised in that the detection of high performance liquid chromatography described in step (2) is mended
The wavelength of bone fat element is 245nm, and the wavelength for detecting apiolin is 338nm.
5. according to the method for claim 1, it is characterised in that described in step (2) used in high performance liquid chromatography efficiently
The model of liquid chromatograph is the series of Agilent 1100.
6. according to the method for claim 1, it is characterised in that the chromatographic column of high performance liquid chromatography described in step (2)
For C18 posts, its model is 250mm × 4.6mm, 5 μm.
7. according to the method for claim 1, it is characterised in that the detection stream of high performance liquid chromatography described in step (2)
Speed is 1mLmin-1。
8. according to the method for claim 1, it is characterised in that the detection temperature of high performance liquid chromatography described in step (2)
Spend for 30 DEG C.
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| CN109342583A (en) * | 2018-09-29 | 2019-02-15 | 景宁畲族自治县食品药品检验检测中心 | It is a kind of it is small it is fragrant hook in psoralen and flavones content detection method |
| CN115327001A (en) * | 2022-09-27 | 2022-11-11 | 广州科曼生物科技有限公司 | Ficus simplicissima lour formula particle contrast extract and preparation method thereof |
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Application publication date: 20180216 |