CN104330516B - A kind of method of simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content - Google Patents
A kind of method of simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content Download PDFInfo
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- CN104330516B CN104330516B CN201410697800.3A CN201410697800A CN104330516B CN 104330516 B CN104330516 B CN 104330516B CN 201410697800 A CN201410697800 A CN 201410697800A CN 104330516 B CN104330516 B CN 104330516B
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Abstract
The invention discloses a kind of method of simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content, comprise the making of reference substance calibration curve, pre-treatment and the mensuration of Chinese yam testing sample, wherein the pre-treatment of testing sample is: the methanol aqueous solution that is 20%-60% by Chinese yam sample volume fraction carries out ultrasonic extraction 20-40min, after shaking up, get extracting section liquid centrifugal to centrifuge tube, after absorption supernatant, adopt membrane filtration to obtain testing sample solution; The mensuration of described Chinese yam testing sample adopts Liquid Chromatography-Tandem Mass Spectrometry method. The present invention combines chromatogram and mass spectrographic advantage, and high specificity, highly sensitive can reach qualification and the object of quality control, for the quality control of Chinese yam and medicine materical crude slice thereof provides reference frame simultaneously.
Description
Technical field
The present invention relates to a kind of method of simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content.
Background technology
Chinese yam, another name RHIIZOMA DIOSCOREAE from Henan of China, Chinese yam rhizome, be called for short Huai Shan, is the dry rhizome of Dioscoreaceae plant Chinese yam,Be medicine-food two-purpose resource, have tonifying kidney and nourishing stomach, the beneficial lung that promotes the production of body fluid, effect of the puckery essence of kidney tonifying, be Liuwei Dihuang Wan,The main component of the Chinese medicine such as the Pill of Anemarrhena, Phellodendrom and Rehmannia, Diaoxinxue Kang, and be widely used in all kinds of Chinese medicine preparations.2010 editions " Chinese pharmacopoeia " record yam variety, and the quality control of Chinese yam and medicine materical crude slice thereof is only limited to routineThe detection such as moisture, ash content and extract, be unfavorable for Chinese yam application and popularization clinically.
Allantoin and adenosine are one of main components of Chinese yam, adenosine compounds to sugar and fatty metabolism,Nucleic acid and protein synthetic etc. plays important regulating action. Pharmacological research shows, adenosine has diastoleBlood vessel, reduce blood pressure, the physiologically active such as decreasing heart rate, inhibition platelet aggregation, lax vascular smooth muscle,Also have and trigger or mediate IP, alleviate the application of the Cardioprotective such as reperfusion injury; Allantoin toolThere is the close phase of effect of repairing epithelial tissue and promote the effects such as skin ulcer and wound healing myogenic and Chinese yamClose. Therefore allantoin and adenosine have great effect in the quality control of Chinese yam and medicine materical crude slice thereof.
The assay of allantoin mainly concentrates on thin-layered chromatography, infra-red chromatography, capillary electricity at presentSwimming method, high performance liquid chromatography etc., but in Chinese yam, be rich in polysaccharide, starchy material, easily disturb orderThe mensuration of mark composition, therefore in current detection method, the pre-treatment of testing sample is more complicated, and specificity is poor,And sensitivity is low.
Summary of the invention
For the problems referred to above, the object of the present invention is to provide a kind of Liquid Chromatography-Tandem Mass Spectrometry that adopts to separateAnd the method for Fast Measurement allantoin in dried rhizoma of Dioscorea opposita and adenosine content, high specificity, highly sensitive, can be simultaneouslyReach the object of qualification and quality control, for the quality control of Chinese yam and medicine materical crude slice thereof provides reference frame.
Technical scheme provided by the invention is:
Measure a method for allantoin in dried rhizoma of Dioscorea opposita and adenosine content simultaneously, comprise reference substance calibration curvePre-treatment and the mensuration of making, Chinese yam testing sample, wherein the pre-treatment of testing sample is: by Chinese yam sampleThe methanol aqueous solution that product are 20%-60% by volume fraction carries out ultrasonic extraction 20-40min, after shaking up, gets portionDivide extract centrifugal to centrifuge tube, after absorption supernatant, adopt membrane filtration to obtain testing sample solution; InstituteThe mensuration of stating Chinese yam testing sample adopts Liquid Chromatography-Tandem Mass Spectrometry method.
Preferably, described time, measure the method for allantoin in dried rhizoma of Dioscorea opposita and adenosine content, wherein,
The making of calibration curve: allantoin and adenosine reference substance by drying under reduced pressure to weight, add and treatSurveying the identical methanol aqueous solution of concentration that sample pre-treatments uses, to prepare the mixed mark of reference substance of variable concentrations moltenLiquid, adopts Liquid Chromatography-Tandem Mass Spectrometry method to measure, and wherein allantoin adopts positive ion mode, glandGlycosides adopts negative ion mode, taking the chromatographic peak area of allantoin or adenosine as ordinate, and its corresponding concentrationFor abscissa carries out linear regression production standard curve;
The mensuration of Chinese yam testing sample: by testing sample through pre-treatment, by Liquid Chromatography-Tandem Mass Spectrometry sideMethod is measured, and wherein allantoin adopts positive ion mode, and adenosine adopts negative ion mode, draws allantoisThe chromatographic peak area of element or adenosine, substitution calibration curve calculates allantoin and gland in Chinese yam testing sampleThe content of glycosides.
Preferably, described time, measure the method for allantoin in dried rhizoma of Dioscorea opposita and adenosine content, described liquid phaseThe chromatographic condition that chromatogram tandem mass spectrometry is measured comprises: chromatographic column: PursuitC182.0*100mm,3.0 μ m; Column temperature: 30 DEG C; Mobile phase is first alcohol and water, isocratic elution, and be 8min analysis time, itsDescribed in mobile phase volume than for methyl alcohol: water=10:90, flow velocity is 0.20ml/min; Sample size: 3 μ l.
Preferably, described time, measure the method for allantoin in dried rhizoma of Dioscorea opposita and adenosine content, described liquid phaseThe mass spectrum condition that chromatogram tandem mass spectrometry is measured comprises: electric spray ion source (ESI); Detector capillaryVoltage 1.3kV; Electron spray voltage 5kV; 50 DEG C of ion source temperatures; 300 DEG C of desolventizing temperature degree; DryAtmospheric pressure 20psi; Atomization gas pressure 50psi; Reaction of high order monitoring (MRM) pattern is measured; AdoptPositive and negative ion switched scan pattern is tested, and wherein allantoin adopts positive ion mode, monitoring female fromSon is m/z156.7, and daughter ion is 113.6 and 96.6, and capillary voltage is-45V that collision energy respectivelyFor 10.5V and 13.0V, adenosine adopts negative ion mode, and monitoring parent ion is m/z268.2, daughter ionBe 135.9 and 252.7, capillary voltage is 55V; Collision energy is respectively 16.0V and 5.0V.
Preferably, described time, measure the method for allantoin in dried rhizoma of Dioscorea opposita and adenosine content, described to be measuredIn the pre-treatment of sample, every 1 Keshan medicine sample adds the methanol aqueous solution that 20ml volume fraction is 40% and entersThe ultrasonic extraction of row.
Preferably, described time, measure the method for allantoin in dried rhizoma of Dioscorea opposita and adenosine content, described ultrasonicExtraction time is 1 time, and ultrasonic extraction time is 30min.
Preferably, described time, measure the method for allantoin in dried rhizoma of Dioscorea opposita and adenosine content, described centrifugalRotating speed be 10000r/min, centrifugation time is 10min.
Preferably, described time, measure the method for allantoin in dried rhizoma of Dioscorea opposita and adenosine content, described filter membraneBe filtered into 0.22 μ m filtering with microporous membrane.
The method of simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content of the present invention, by liquid chromatogram withMass spectrum series connection is used, and has that separating power is strong, a high specificity, highly sensitive feature, can reach simultaneouslyThe object of qualification and quality control, compares high performance liquid chromatography, the pre-treatment of testing sample of the present inventionMethod is simple, quick, has following beneficial effect:
(1) pre-treatment of testing sample of the present invention adopts ultrasonic wave to extract, simple to operate, the time is short,While adopting methanol aqueous solution that 20ml volume fraction is 40% as extractant, use once ultrasonic extraction30min extraction efficiency is the highest;
(2) the present invention by the testing sample solution after ultrasonic under the rotating speed of 10000r/min centrifugal 10Min, fully precipitates the large molecules such as starch in Chinese yam, has improved testing sample solution and has carried out liquid chromatogramAnti-interference when tandem mass spectrum is measured;
(3) adopt 0.22 μ m filtering with microporous membrane, filter fine particle, prevent from entering pillar and cause stifledPlug and damage mass spectrum;
(4) assay method of the present invention is highly sensitive, and the detectability that detects allantoin and adenosine is respectively0.002,0.006 μ g/ml, quantitative limit is respectively 0.015,0.080 μ g/ml.
Brief description of the drawings
Fig. 1 is that reaction monitoring pattern of the present invention (MRM) is measured the allantoin standard items chromatogram obtainingFigure;
Fig. 2 is that multiple-reaction monitoring pattern of the present invention (MRM) is measured the adenosine standard items chromatogram obtainingFigure;
Fig. 3 is that multiple-reaction monitoring pattern of the present invention (MRM) is measured gland in the yam extract obtainingGlycosides and allantoin chromatogram;
Fig. 4 is allantoin calibration curve of the present invention;
Fig. 5 is adenosine calibration curve of the present invention.
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is described in further detail, to make those skilled in the art's referenceDescription word can be implemented according to this.
The invention provides a kind of method of simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content, comprise following stepRapid:
(1) making of calibration curve: allantoin and adenosine reference substance by drying under reduced pressure to weight, addVolume fraction is that 40% methanol aqueous solution is mixed with that every 1ml contains 150.0 μ g allantoins and every 1ml containsThere is the mixed mark solution of 20.0 μ g adenosines, be diluted to as required the mixed mark solution of variable concentrations, it is first logicalCross liquid chromatogram and separate allantoin and adenosine, then pass through mass spectroscopy, wherein allantoin adopts cation mouldFormula, adenosine adopts negative ion mode, taking the chromatographic peak area Y of allantoin or adenosine as ordinate, its phaseCorresponding concentration X is that abscissa carries out linear regression production standard curve, and investigates linear relationship, respectivelyArrive calibration curve as Fig. 4 and Fig. 5, calibration curve, the range of linearity, coefficient correlation, quantitative limit and detectionLimit is in table 1;
Calibration curve, the range of linearity, coefficient correlation, quantitative limit and the detectability of table 1 allantoin and adenosine
(2) pre-treatment of Chinese yam testing sample and mensuration: get Chinese yam sample powder 1g in 25ml volumetric flaskIn, adding 20ml volume fraction is 40% methanol aqueous solution, once after ultrasonic extraction 30min, is settled to25ml, shakes up, and gets extracting section liquid to centrifuge tube, and centrifugal rotational speed is 10000r/min, and centrifugation time is10min, draws supernatant and obtains testing sample solution through 0.22 μ m filtering with microporous membrane, is first passed throughLiquid chromatogram separates allantoin and adenosine wherein, then passes through mass spectroscopy, wherein allantoin adopt just fromSubpattern, adenosine adopts negative ion mode, draws the chromatographic peak area of allantoin or adenosine, substitution standardCurve calculation draws the content of allantoin and adenosine in Chinese yam testing sample.
(3) condition determination:
Chromatographic condition: PursuitC182.0*100mm, 3.0 μ m; Column temperature: 30 DEG C; Mobile phase is firstAlcohol and water, isocratic elution 8min, wherein said mobile phase volume is than being methyl alcohol: water=10:90, flow velocity is0.20ml/min; Sample size: 3 μ l;
Mass spectrum condition: electric spray ion source (ESI); Detector capillary voltage 1.3kV; Electron spray electricityPress 5kV; 50 DEG C of ion source temperatures; 300 DEG C of desolventizing temperature degree; Dry gas pressure 20psi; Atomization gasPressure 50psi; Reaction of high order monitoring pattern is measured; Adopt positive and negative ion switched scan pattern to test,Wherein allantoin adopts positive ion mode, and allantoin parent ion is m/z156.7, and daughter ion is 113.6 Hes96.6, go bunch voltage to be-45V, collision energy is respectively 10.5V and 13.0V, and adenosine adopts anion mouldFormula, adenosine parent ion is m/z268.2, and daughter ion is 135.9 and 252.7, and removing bunch voltage is 55V; TouchHit energy and be respectively 16.0V and 5.0V.
(4) data analysis:
The standard items allantoin (RT=1.41) that multiple-reaction monitoring pattern (MRM) is measured and adenosine (RT=5.22)Chromatogram, as depicted in figs. 1 and 2; In the yam extract that multiple-reaction monitoring pattern (MRM) is measuredThe chromatogram of allantoin (RT=1.41) and adenosine (RT=5.22), as shown in Figure 3. By with standardAllantoin and adenosine contrast can differentiate allantoin and the adenosine in yam extract.
Known according to table 1, Fig. 1, Fig. 2 and Fig. 3, the Liquid Chromatography-Tandem Mass Spectrometry adopting detects allantoisElement is good with the chromatographic peak separating degree of adenosine, high specificity, and its corresponding concentration of chromatographic peak area itBetween there is good linear correlation. Allantoin, the adenosine content of the present embodiment Chinese yam sample are respectively3.97mg/g、0.105mg/g。
(4) recovery test
Test adopts standard addition method to carry out rate of recovery experiment, takes the Chinese yam sample of 9 parts of known content1.0g, adds respectively high, medium and low different allantoin, the adenosine standard items of measuring, according to pre-treating methodPrepare testing sample solution, measure and calculate recovery rate by above-mentioned chromatographic condition sample introduction, in table 2. AllantoisThe average recovery of element is 97.4%～103.3%, and the average recovery of adenosine is 98.1～103.1. Test knotFruit shows that set up method has higher standard for the content of measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine simultaneouslyExactness.
The average recovery of table 2 allantoin and adenosine
Although embodiment of the present invention are open as above, it is not restricted to description and enforcement sideListed utilization in formula, it can be applied to various applicable the field of the invention completely, for being familiar with abilityThe personnel in territory, can easily realize other amendment, therefore do not deviate from claim and etc. homotypeEnclose under limited universal, the present invention is not limited to specific details and illustrates here and the figure describingExample.
Claims (5)
1. a method of simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content, is characterized in that, it is right to compriseAccording to the making of product calibration curve, pre-treatment and the mensuration of Chinese yam testing sample, the wherein front place of testing sampleReason is: the methanol aqueous solution that is 20%-60% by volume fraction by Chinese yam sample carries out ultrasonic extraction20-40min, gets extracting section liquid centrifugal to centrifuge tube after shaking up, after absorption supernatant, adopt membrane filtrationObtain testing sample solution; The mensuration of described Chinese yam testing sample adopts Liquid Chromatography-Tandem Mass Spectrometry method, itsIn,
The making of calibration curve: allantoin and adenosine reference substance by drying under reduced pressure to weight, add and treatSurveying the identical methanol aqueous solution of concentration that sample pre-treatments uses, to prepare the mixed mark of reference substance of variable concentrations moltenLiquid, adopts Liquid Chromatography-Tandem Mass Spectrometry method to measure, and wherein allantoin adopts positive ion mode, glandGlycosides adopts negative ion mode, taking the chromatographic peak area of allantoin or adenosine as ordinate, and its corresponding concentrationFor abscissa carries out linear regression production standard curve;
The mensuration of Chinese yam testing sample: by testing sample through pre-treatment, by Liquid Chromatography-Tandem Mass Spectrometry sideMethod is measured, and wherein allantoin adopts positive ion mode, and adenosine adopts negative ion mode, draws allantoisThe chromatographic peak area of element or adenosine, substitution calibration curve calculates allantoin and gland in Chinese yam testing sampleThe content of glycosides;
The chromatographic condition that described Liquid Chromatography-Tandem Mass Spectrometry method is measured comprises: chromatographic column: PursuitC182.0*100mm, 3.0 μ m; Column temperature: 30 DEG C; Mobile phase is first alcohol and water, isocratic elution, analysis timeFor 8min, wherein said mobile phase volume is than being methyl alcohol: water=10:90, and flow velocity is 0.20ml/min; Sample introductionAmount: 3 μ l;
The mass spectrum condition that described Liquid Chromatography-Tandem Mass Spectrometry method is measured comprises: electric spray ion source (ESI);Detector capillary voltage 1.3kV; Electron spray voltage 5kV; 50 DEG C of ion source temperatures; Desolventizing temperatureSpend 300 DEG C; Dry gas pressure 20psi; Atomization gas pressure 50psi; Reaction of high order monitoring (MRM) mouldFormula is measured; Adopt positive and negative ion switched scan pattern to test, wherein allantoin adopts cation mouldFormula, monitoring parent ion is m/z156.7, and daughter ion is 113.6 and 96.6, and capillary voltage is-45V,Collision energy is respectively 10.5V and 13.0V, and adenosine adopts negative ion mode, and monitoring parent ion is m/z268.2, daughter ion is 135.9 and 252.7, and capillary voltage is 55V; Collision energy is respectively 16.0VAnd 5.0V.
2. the method for simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content as claimed in claim 1, its spyLevy and be, in the pre-treatment of described testing sample, every 1 Keshan medicine sample adds 20ml volume fraction and is40% methanol aqueous solution carries out ultrasonic extraction.
3. the method for simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content as claimed in claim 1, its spyLevy and be, described ultrasonic extraction time is 1 time, and ultrasonic extraction time is 30min.
4. the method for simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content as claimed in claim 1, its spyLevy and be, described centrifugal rotating speed is 10000r/min, and centrifugation time is 10min.
5. the method for simultaneously measuring allantoin in dried rhizoma of Dioscorea opposita and adenosine content as claimed in claim 1, its spyLevy and be, described membrane filtration is 0.22 μ m filtering with microporous membrane.
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| CN108414638A (en) * | 2018-03-24 | 2018-08-17 | 厦门市诚安毅科技有限公司 | The detection method of active constituent in a kind of quick detection common yam rhizome powder |
| CN109839470A (en) * | 2019-02-25 | 2019-06-04 | 亚宝药业贵阳制药有限公司 | A kind of thin-layer identification method of invigorating the spleen gel products |
| CN111559979A (en) * | 2020-06-12 | 2020-08-21 | 劲牌有限公司 | Method for preparing allantoin by Prep-HPLC method |
| CN115015452B (en) * | 2022-06-07 | 2023-11-17 | 山东宏济堂制药集团股份有限公司 | Method for measuring content of allantoin and adenosine in Chinese yam by adopting one-measurement-multiple-evaluation method |
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| CN1958575A (en) * | 2006-11-03 | 2007-05-09 | 威海清华紫光科技开发有限公司 | Method for extracting polysaccharide and allantoin from Chinese yam |
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