CN106018580A - Method for measuring concentration of skullcapflavone II in plasma - Google Patents
Method for measuring concentration of skullcapflavone II in plasma Download PDFInfo
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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
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Abstract
The invention discloses a method for measuring concentration of 5,2'-dyhydroxyl,6,7,8,5'-tetramethoxyflavone (skullcapflavone II) in plasma. A liquid chromatography-mass spectrometry system is used for carrying out measuring, a sample to be measured is firstly taken, a certain amount of organic solvent is added for medicine liquid-liquid extraction, after pretreatment, chromatographic column separation is carried out, and a mass spectrometry detector is used for carrying out detection. According to the method, quickness and accuracy are achieved, sensitivity is high, and operation is easy and convenient; the method is suitable for measuring the concentration of skullcapflavone II in the plasma.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of measure in blood plasma 5,2`-dihydroxy, the method for 6,7,8,5`-tetramethoxy flavones (skullcapflavone II) concentration.
Background technology
Skullcapflavone-II, i.e. 5,2`-dihydroxy, 6,7,8,5`-tetramethoxy flavones, are the Novel flavonoids of isolated from medicinal plants Radix Scutellariae.SKullcapflavone-II has anti-inflammatory, viral and immunoregulatory effect.Additionally, many compositions have antipruritic, regulation metabolism disorder, neuroprotective, promotion angiogenesis, the anticancer and effect of AntiHIV1 RT activity.Skullcapflavone-II has various biological characteristic, as Cytotoxic supposition acts on, and the impact on mastocyte histamine release, raise inhibitor and asthma Airway inflammatory response by trypsin suppression activator of plasminogen 1 type.
Its structural formula is as follows:
The most not yet document about Skullcapflavone-II in vivoassay method is reported.The present invention establishes Skullcapflavone-II assay method in beagle dog body, by the method, the drug metabolism situation of Skullcapflavone-II is carried out exploratory study.
Summary of the invention
The technical problem to be solved is to set up a kind of precise and high efficiency to measure the method for skullcapflavone-II concentration in blood plasma.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows:
A kind of measuring the method for skullcapflavone-II concentration in blood plasma, detect its concentration through LC-MS system after plasma sample is preprocessed, concrete grammar comprises the steps:
(1) plasma sample pretreatment: take plasma sample, adds internal standard neobavaisoflavone, adds a certain amount of ethyl acetate and carry out organic solvent extraction, takes supernatant after 40 DEG C of nitrogen dry up, with sample introduction analysis after flowing phased soln；
(2) step (1) pretreated plasma sample is carried out liquid chromatograph separation: in chromatographic condition, chromatographic column is Agilent ZORBAX SB-C18Post；Flowing phase: volume ratio is acetonitrile and the water of 80: 20；Flow velocity: 0.2mL min-1；Column temperature: 40 DEG C；Type of elution is isocratic elution；
(3) mass spectroscopy, condition is:
Ion source: electro-spray ionization ionization source ESI；Ion detection mode: multiple-reaction monitoring MRM；Ion polarity: cation；Skullcapflavone-II collides voltage 23eV；Internal standard neobavaisoflavone collision voltage 29eV；Capillary voltage 4.5kV；Gas curtain gas Curtain gas:20psi；Ion source Gas1:70psi；Ion source Gas2:80psi；Desolventizing temperature: 400 DEG C；CXP:9V；Collision gas CAD:10V；DP:100V；EP:10V.The ion channel of detection object: skullcapflavone-II m/z 375.1 → 345.1, internal standard neobavaisoflavone m/z 323.3 → 255.1
(4) calculate: use internal standard method, substitute into the concentration of standard curve Equation for Calculating skullcapflavone-II with the peak area ratio of skullcapflavone-II and internal standard neobavaisoflavone.
Specifically, in step (1), take plasma sample 500 μ L, add 100ng mL-1Internal standard neobavaisoflavone 20 μ L, adds 4mL ethyl acetate, and after concussion 3min, 3500 × g is centrifuged 10min, takes supernatant 3.5mL after 40 DEG C of nitrogen dry up, after 100 μ L flowing phased solns, and 10 μ L sample introductions analyses.
In step (2), chromatogram column length is 150mm, and internal diameter is 2.1mm, and packing material size is 5 μm.
Wherein, described blood plasma is to give the blood plasma containing skullcapflavone-II medicine.
In a specific embodiment, described blood plasma is the blood plasma of human or animal.
In a specific embodiment, described blood plasma is dog plasma.Described dog is beagle dog.
Beneficial effect: the inventive method compared with prior art has the advantage that
(1) preprocess method is easy: use organic solvent fluid page extraction, it is adaptable to conventional determining.
(2) specificity is strong: use Agilem ZORBAX SB-C18Chromatographic column as fixing phase, acetonitrile and water mixed liquor as flowing phase, isocratic elution, Skullcapflavone-II retention time is about 3.09min, internal standard neobavaisoflavone retention time is that about 2.88min, 4.5min complete to measure, and endogenous material does not disturb the mensuration of the two.
(3) highly sensitive: blood plasma is minimum is quantitatively limited to 1ng mL-1。
(4) the inventive method is quick, accurate, highly sensitive, easy and simple to handle, and the determination of plasma concentration for Skullcapflavone-II provides foundation, has the prospect of new drug development.The plasma standard curve linear scope of this method is 1～4000ng mL-1, in a few days it is respectively less than 10% with day to day precision RSD.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of dog blank plasma；
Fig. 2 is that dog blank plasma adds Skullcapflavone-II and the mass spectrum of internal standard reference substance；
Fig. 3 be after dog gives Skullcapflavone-II plasma sample add internal standard reference substance mass spectrum；
Note: figure intermediate ion passage 1 is Skullcapflavone-II, [M-H]+, m/z 375.1 → 345.1, retention time is about 3.09min；Ion selector channel 2 is internal standard neobavaisoflavone, [M-H]+, m/z 323.3 → 255.1, retention time is about 2.88min.
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that the content described by embodiment is merely to illustrate the present invention, and should be also without limitation on the present invention described in detail in claims.
Embodiment: the mensuration of Skullcapflavone-II concentration in dog plasma
One, experiment material and instrument
Skullcapflavone-II: provided (Nanjing, China) by damp bright Pharmaceutical Group；Neobavaisoflavone (internal standard): be purchased from national drug biological products assay institute and buy (Beijing, China)；Test water: ultra-pure water；Methanol: chromatographically pure (Merck Company).
Agilent ZORBAX SB-C18Post (150mm × 2.1mm I.D., 5 μm, Agilent Technologies, Wilmington, DE, USA)；API4000 LC-MS/MS triple level Four bar mass spectrograph (American AB company limited), chromatographic work station is Analyst 1.4.2；CPA225D electronic analytical balance (Sai Duolisi company limited of Germany)；WH-2 miniature vortex mixed instrument (Shanghai Hu Xi analytical tool factory)；Milli-Q system water purification machine (micropore, Bedford, MA, USA)；Biofuge PrimoR high-speed refrigerated centrifuge (Heraeus company of Germany).
Two, liquid matter condition
1. liquid phase chromatogram condition
Chromatographic column: Agilent ZORBAX SB-C18 post (150mm × 2.1mm I.D., 5 μm, Agilent Technologies, Wilmington, DE, USA)；Flowing phase: acetonitrile and aqueous solution (80: 20, v/v)；Flow velocity: 0.2mL min-1；Column temperature: 40 DEG C.
2. Mass Spectrometry Conditions
Ion source: electro-spray ionization ionization source ESI；Ion detection mode: multiple-reaction monitoring MRM；Ion polarity: cation；Skullcapflavone-II collides voltage 23eV；Internal standard neobavaisoflavone collision voltage 29eV；Capillary voltage 4.5kV；Curtain gas:20；Gasl:70；Gas2:80；Desolventizing temperature: 400 DEG C；CXP:9；CAD:10；DP:100；EP:10.The ion channel of detection object: skullcapflavone-II m/z 375.1 → 345.1, internal standard neobavaisoflavone m/z 323.3 → 255.1.
Three, experimentation:
The preparation of 1.Skullcapflavone-II standard solution:
Precision weighs Skullcapflavone-II 10.08mg, is placed in 10mL volumetric flask, adds methanol and dissolves and be settled to scale, shakes up, obtain 1.008mg mL-1The storing solution of Skullcapflavone-II.Precision measures appropriate storing solution and dilutes successively with methanol, is made into concentration and is respectively 1,5,10,50,100,500,1000,4000ng mL-1Skullcapflavone-II standard solution.
The collection of 2.beagle dog plasma sample and process:
Beagle dog intravenous injection Skullcapflavone-II solution, dosage is 5mg kg-1, in injection before and injection after 5,10,15,30,45min and 1,1.5,2,3,4,6,8,12h venous blood collection 1mL, injection calparine pipe in, 3000rpm is centrifuged 10min, prepares blood plasma.Take beagle dog plasma 500 μ L, add 100ng mL-1Internal standard neobavaisoflavone 20 μ L, adds 4mL ethyl acetate, and after concussion 3min, 3500 × g is centrifuged 10min, takes supernatant 3.5mL after 40 DEG C of nitrogen dry up, after 100 μ L flowing phased solns, and 10 μ L sample introductions analyses.
3. specificity:
Take the beagle dog blank plasma 500 μ L being not given to Skullcapflavone-II, adding 4mL ethyl acetate, after concussion 3min, 3500 × g is centrifuged 10min, takes supernatant 3.5mL after 40 DEG C of nitrogen dry up, after 100 μ L flowing phased solns, 10 μ L sample introductions are analyzed.
Taking centrifuge tube number to prop up, add Skullcapflavone-II standard solution 10 μ L, 40 DEG C of nitrogen dry up, add the beagle dog blank plasma 500 μ L being not given to Skullcapflavone-II, vortex 30s mixes, and adds neobavaisoflavone (internal standard, 100ng.mL-1) 10 μ L, adding 4mL ethyl acetate, after concussion 3min, 3500 × g is centrifuged 10min, takes supernatant 3.5mL after 40 DEG C of nitrogen dry up, after 100 μ L flowing phased solns, 10 μ L sample introductions analyses.
Result shows, under the conditions of the LC-MS/MS that this experiment is used, detected components is interfered by plasma sample without miscellaneous peak, and Skullcapflavone-II retention time is at about 3.09min, and internal standard Corylin. retention time is at about 2.88min.Skullcapflavone-II and internal standard do not interfere with each other, and peak shape is good, and baseline is steady.
4. linear test
Beagle dog plasma standard curve: take centrifuge tube number and prop up, precision adds the Skullcapflavone-II standard solution 10 μ L of variable concentrations respectively, and 40 DEG C of nitrogen dry up, and adds the blank plasma 500 μ L vortex 30s mixing being not given to Skullcapflavone-II, it is made into the concentration containing Skullcapflavone-II and is respectively 0.001,0.005,0.01,0.05,0.1,0.5, the plasma containing drug of Isosorbide-5-Nitrae μ g/mL.Add neobavaisoflavone (internal standard, 100ng mL-1) 10 μ L, adding 4mL ethyl acetate, after concussion 3min, 3500 × g is centrifuged 10min, takes supernatant 3.5mL after 40 DEG C of nitrogen dry up, after 100 μ L flowing phased solns, 10 μ L sample introductions analyses.Calculate Skullcapflavone-II and the ratio Y of internal standard peak area, with peak area ratio Y, blood drug level X is made regression Calculation, obtaining regression equation: Y=(0.00171 ± 0.00035) X+ (0.00067 ± 0.00029) (n=5), Skullcapflavone-II plasma concentration is at 1～4000ng mL-1In the range of linear relationship good.Minimum quantitatively it is limited to 1ng mL-1。
5. accuracy and precision
Containing Skullcapflavone-II concentration according to the preparation of beagle dog plasma standard curve method is 0.002,0.02and2 μ g/mL's is basic, normal, high containing Skullcapflavone-II plasma sample, processes respectively by " process of beagle dog plasma sample " processing method.Do 3 days continuously, every day, each concentration respectively made 5 parts of samples, calculate Skullcapflavone-II peak area As and the ratio f of internal standard peak area Ai, substitute in the plasma standard curve on the same day and try to achieve Skullcapflavone-II measured concentration, calculated in a few days by measured concentration, day to day precision and relative standard deviation (RSD), measured concentration and the ratio adding concentration are accuracy.Result shows, is in a few days respectively less than 10% with day to day precision RSD, and accuracy meets the requirements.
6. measurement result
Above-mentioned 6 were only given Skullcapflavone-II content in the beagle dog plasma of Skullcapflavone-II (5min after administration) is respectively 2.17,2.04,2.35,2.23,2.19,2.24 μ g/mL.
The present invention is by using Agilent ZORBAX SB-C18Chromatographic column is as fixing phase, the mixed liquor of acetonitrile and water is as flowing phase, isocratic elution, Skullcapflavone-II retention time is about 3.09min, internal standard neobavaisoflavone retention time is about 2.88min, 4.5min completes to measure, and endogenous material does not disturb the mensuration of the two, and preprocess method is easy simultaneously；Use organic solvent fluid page extraction, it is adaptable to conventional determining；Highly sensitive, blood plasma is minimum is quantitatively limited to 1ng mL-1, and the inventive method is quick, accurate, highly sensitive, easy and simple to handle, the determination of plasma concentration for Skullcapflavone-II provides foundation, has the prospect of new drug development.The plasma standard curve linear scope of this method is 1～4000ng mL-1, in a few days it is respectively less than 10% with day to day precision RSD.
Claims (6)
1. one kind measures the method for skullcapflavone II concentration in blood plasma, it is characterised in that plasma sample is preprocessed
Detecting its concentration by LC-MS system, concrete grammar comprises the steps:
(1) plasma sample pretreatment: take plasma sample, adds internal standard neobavaisoflavone, adds a certain amount of second
Acetoacetic ester carries out organic solvent extraction, takes after supernatant nitrogen dries up, obtains pretreated blood plasma sample with flowing phased soln
Product；
(2) step (1) pretreated plasma sample being carried out liquid chromatograph separation: in chromatographic condition, chromatographic column is
Agilent ZORBAX SB-C18Post；Flowing is mutually for acetonitrile that volume ratio is 80: 20 and water；Flow velocity is 0.2-0.5
mL·min-1；Column temperature is 40 DEG C；Type of elution is isocratic elution；
(3) mass spectroscopy, condition is:
Ion source: electro-spray ionization ionization source ESI；Ion detection mode: multiple-reaction monitoring MRM；Ion polarity:
Cation；Skullcapflavone-II collides voltage 23eV；Internal standard neobavaisoflavone collision voltage 29eV；Hair
Tubule voltage 4.5kV；Gas curtain gas Curtain gas:20psi；Ion source Gas1:70psi；Ion source Gas2:80psi；
Desolventizing temperature: 400 DEG C；CXP:9V；CAD:10V；DP:100V；EP:10V, detection object from
Subchannel: skullcapflavone-II m/z 375.1 → 345.1, internal standard neobavaisoflavone m/z 323.3 → 255.1
(4) calculate: use internal standard method, with skullcapflavone-II and the peak area ratio of internal standard neobavaisoflavone
Value substitutes into the concentration of standard curve Equation for Calculating skullcapflavone-II.
The method of skullcapflavone-II concentration in mensuration blood plasma the most according to claim 1, it is characterised in that
In step (1), take plasma sample 500 μ L, add 100ng mL-1Internal standard neobavaisoflavone 20 μ L, adds
4mL ethyl acetate, after concussion 3min, 3500 × g is centrifuged 10min, takes supernatant 3.5mL after 40 DEG C of nitrogen dry up,
After 100 μ L flowing phased solns, 10 μ L sample introductions are analyzed.
The method of skullcapflavone-II concentration in mensuration blood plasma the most according to claim 1, it is characterised in that
In step (2), chromatogram column length is 150mm, and internal diameter is 2.1mm, and packing material size is 5 μm.
The method of skullcapflavone-II concentration in mensuration blood plasma the most according to claim 1, it is characterised in that
Described blood plasma is to give the blood plasma containing skullcapflavone-II medicine.
The method of skullcapflavone-II concentration in mensuration blood plasma the most according to claim 1, it is characterised in that
Described blood plasma is the blood plasma of human or animal.
The method of skullcapflavone-II concentration in mensuration blood plasma the most according to claim 1, it is characterised in that
Described blood plasma is dog plasma.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444301A (en) * | 2018-12-18 | 2019-03-08 | 江苏省中医院 | A kind of method of general reed Ka Bili concentration in measurement blood plasma |
| CN109633039A (en) * | 2019-02-03 | 2019-04-16 | 北京中医药大学 | The detection method of hydroxyl polymethoxyflavone compound and its metabolite in biological sample |
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| CN1395573A (en) * | 2000-01-11 | 2003-02-05 | 比奥雷克斯健康有限公司 | Extraction of flavonoids |
| KR20130120849A (en) * | 2012-04-26 | 2013-11-05 | 한국생명공학연구원 | A pharmaceutical compositon containing skullcapflavone ii or pharmaceutically acceptable salt thereof for the prevention or treatment of asthma |
-
2016
- 2016-05-11 CN CN201610308527.XA patent/CN106018580B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1395573A (en) * | 2000-01-11 | 2003-02-05 | 比奥雷克斯健康有限公司 | Extraction of flavonoids |
| KR20130120849A (en) * | 2012-04-26 | 2013-11-05 | 한국생명공학연구원 | A pharmaceutical compositon containing skullcapflavone ii or pharmaceutically acceptable salt thereof for the prevention or treatment of asthma |
Non-Patent Citations (3)
| Title |
|---|
| HA-YOUNG JANG ET AL.: "Skullcapflavone II inhibits ovalbumin-induced airway inflammation in a mouse model of asthma", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 * |
| 陈佩东 等: "黄芩中黄酮类成分的分离鉴定及其体外对凝血系统的影响", 《中草药》 * |
| 黄勇 等: "UPLC-MS/MS法同时测定家兔血浆中6个黄酮类成分及其药代动力学研究", 《药物分析杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444301A (en) * | 2018-12-18 | 2019-03-08 | 江苏省中医院 | A kind of method of general reed Ka Bili concentration in measurement blood plasma |
| CN109633039A (en) * | 2019-02-03 | 2019-04-16 | 北京中医药大学 | The detection method of hydroxyl polymethoxyflavone compound and its metabolite in biological sample |
| CN109633039B (en) * | 2019-02-03 | 2021-09-10 | 北京中医药大学 | Method for detecting hydroxyl polymethoxylated flavone compound and metabolite thereof in biological sample |
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