CN106018580B - A kind of method of skullcapflavone II concentration in measure blood plasma - Google Patents
A kind of method of skullcapflavone II concentration in measure blood plasma Download PDFInfo
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- CN106018580B CN106018580B CN201610308527.XA CN201610308527A CN106018580B CN 106018580 B CN106018580 B CN 106018580B CN 201610308527 A CN201610308527 A CN 201610308527A CN 106018580 B CN106018580 B CN 106018580B
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- 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
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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
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Abstract
The invention discloses one kind determine blood plasma in 5,2` dihydroxy, 6, the method of 7,8,5` tetramethoxy flavones (skullcapflavone II) concentration, using LC-MS system measurement, testing sample is first taken, a certain amount of organic solvent is added and carries out medicine liquid-liquid extraction, after pretreatment, through chromatogram post separation, detected with mass detector, the inventive method is quick, accurate, high sensitivity, easy to operate, is suitable for determining the concentration of skullcapflavone II in blood plasma.
Description
Technical field
The invention belongs to technical field of biological, and in particular to 5,2`- dihydroxy in one kind measure blood plasma, 6,7,8,5
The method of `- tetramethoxy flavones (skullcapflavone II) concentration.
Background technology
Skullcapflavone-II, i.e. 5,2`- dihydroxy, 6,7,8,5`- tetramethoxy flavones, it is yellow from medicinal plant
Isolated Novel flavonoids in a kind of reed mentioned in ancient books.SKullcapflavone-II has to anti-inflammatory, virus and immunoregulatory
Effect.In addition, many compositions have antipruritic, regulation metabolic disorder, neuroprotection, the work for promoting angiogenesis, anticancer and AntiHIV1 RT activity
With.Skullcapflavone-II has various biological characteristic, and such as the hypothesis effect of cytotoxicity, mast cell histamine is released
The influence put, the type of activator of plasminogen 1 is suppressed by trypsase and raises inhibitor and the reaction of anti-airway inflammation.
Its structural formula is as follows：
Document report not yet on Skullcapflavone-II in vivoassay methods at present.The present invention establishes
Assay methods of the Skullcapflavone-II in beagle dog bodies, the medicine by this method to Skullcapflavone-II
Thing metabolic condition carries out pilot study.
The content of the invention
The technical problems to be solved by the invention are to establish skullcapflavone- in a kind of precise and high efficiency measure blood plasma
The method of II concentration.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of method of skullcapflavone-II concentration in measure blood plasma, join after plasma sample is preprocessed through liquid matter
With its concentration of system detectio, specific method comprises the following steps：
(1) plasma sample pre-processes：Plasma sample is taken, internal standard neobavaisoflavone is added, adds a certain amount of acetic acid
Ethyl ester carries out organic solvent extraction, takes supernatant after 40 DEG C of nitrogen dry up, and is analyzed with the laggard sample of flowing phased soln；
(2) the pretreated plasma sample of step (1) is subjected to liquid chromatogram separation：Chromatographic column is in chromatographic condition
Agilent ZORBAX SB-C18Post；Mobile phase：The acetonitrile and water that volume ratio is 80: 20；Flow velocity：0.2mL·min-1；Post
Temperature：40℃；Type of elution is isocratic elution；
(3) mass spectroscopy, condition are：
Ion gun：Electro-spray ionization ionization source ESI；Ion detection mode：Multiple-reaction monitoring MRM；Ion polarity：Just from
Son；Skullcapflavone-II collision voltages 23eV；Internal standard neobavaisoflavone collision voltage 29eV；Capillary voltage
4.5kV；Gas curtain gas Curtain gas：20psi；Ion gun Gas1：70psi；Ion gun Gas2：80psi；Desolventizing temperature：
400℃；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：Using internal standard method, with skullcapflavone-II and the peak area ratio of internal standard neobavaisoflavone
Value substitutes into the concentration that calibration curve equation calculates skullcapflavone-II.
Specifically, in step (1), the μ L of plasma sample 500 are taken, add 100ngmL-1The μ of internal standard neobavaisoflavone 20
L, 4mL ethyl acetate is added, shake 3500 × g centrifugation 10min after 3min, take supernatant 3.5mL to be used after 40 DEG C of nitrogen dry up
After 100 μ L flowing phased solns, the analysis of 10 μ L sample introductions.
In step (2), chromatogram column length 150mm, internal diameter 2.1mm, packing material size are 5 μm.
Wherein, described blood plasma is the blood plasma for giving the medicine containing skullcapflavone-II.
In a specific embodiment, the blood plasma is the blood plasma of human or animal.
In a specific embodiment, the blood plasma is dog plasma.Described dog is beagle dogs.
Beneficial effect：The inventive method has following advantage compared with prior art：
(1) preprocess method is easy：Using organic solvent fluid page extraction, suitable for conventional determining.
(2) specificity is strong：Using Agilem ZORBAX SB-C18Chromatographic column is as stationary phase, the mixed liquor of acetonitrile and water
As mobile phase, isocratic elution, Skullcapflavone-II retention times are 3.09min or so, the different Huang of the new psoralea corylifolia of internal standard
Ketone retention time is 2.88min or so, and 4.5min completes measure, and endogenous material does not disturb the measure of the two.
(3) high sensitivity：Blood plasma is minimum to be quantitatively limited to 1ngmL-1。
(4) the inventive method is quick, accurate, high sensitivity, easy to operate, is that Skullcapflavone-II blood medicine is dense
Degree measure provides foundation, the prospect with 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.
Brief description of the drawings
Fig. 1 is the mass spectrogram of dog blank plasma；
Fig. 2 is the mass spectrogram that dog blank plasma adds Skullcapflavone-II and internal standard reference substance；
Fig. 3 is that dog gives the mass spectrogram that plasma sample after Skullcapflavone-II adds internal standard reference substance；
Note：Figure intermediate ion passage 1 is Skullcapflavone-II, [M-H]+, m/z 375.1 → 345.1, retention time
For 3.09min or so；Ion selector channel 2 is internal standard neobavaisoflavone, [M-H]+, m/z 323.3 → 255.1, retain
Time is 2.88min or so.
Embodiment
According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should be also without limitation on sheet described in detail in claims
Invention.
Embodiment：The measure of Skullcapflavone-II concentration in dog plasma
First, experiment material and instrument
skullcapflavone-II：There is provided (Nanjing, China) by the bright Pharmaceutical Group in pool；Neobavaisoflavone is (interior
Mark)：It is purchased from national drug biological products assay institute purchase (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)；The triple level Four bar mass spectrographs of API4000 LC-MS/MS (American AB Co., Ltd), chromatogram work
Stand as Analyst 1.4.2；CPA225D electronic analytical balances (German Sai Duolisi Co., Ltds)；The miniature vortex mixing of WH-2
Instrument (Shanghai Hu Xi analytical instrument factory)；Milli-Q systems water purification machine (micropore, Bedford, MA, USA)；Biofuge PrimoR
High-speed refrigerated centrifuge (German Heraeus companies).
2nd, liquid matter condition
1. liquid phase chromatogram condition
Chromatographic column：Agilent ZORBAX SB-C18 posts (150mm × 2.1mm I.D., 5 μm, Agilent
Technologies, Wilmington, DE, USA)；Mobile phase：Acetonitrile and the aqueous solution (80: 20, v/v)；Flow velocity：0.2mL·
min-1；Column temperature：40℃.
2. Mass Spectrometry Conditions
Ion gun：Electro-spray ionization ionization source ESI；Ion detection mode：Multiple-reaction monitoring MRM；Ion polarity：Just from
Son；Skullcapflavone-II collision voltages 23eV；Internal standard neobavaisoflavone collision voltage 29eV；Capillary voltage
4.5kV；Curtain gas：20；Gasl：70；Gas2：80；Desolventizing temperature：400℃；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。
3rd, experimentation：
The preparation of 1.Skullcapflavone-II standard liquids：
Precision weighs Skullcapflavone-II 10.08mg, is placed in 10mL volumetric flasks, adds methanol and dissolves and determine
Hold to scale, shake up, produce 1.008mgmL-1Skullcapflavone-II storing solution.Precision measure appropriate storing solution with
Methanol dilutes successively, and it is respectively 1,5,10,50,100,500,1000,4000ngmL to be made into concentration-1's
Skullcapflavone-II standard liquids.
The collection and processing of 2.beagle dog plasma samples：
Beagle dogs are injected intravenously Skullcapflavone-II solution, dosage 5mgkg-1, before injection and note
5,10,15,30,45min and 1,1.5,2,3,4,6,8,12h venous blood collection 1mL after penetrating, inject in calparine pipe, 3000rpm centrifugations
10min, prepare blood plasma.The μ L of beagle dog plasmas 500 are taken, add 100ngmL-1The μ L of internal standard neobavaisoflavone 20, add
4mL ethyl acetate, 3500 × g centrifugation 10min after 3min are shaken, supernatant 3.5mL are taken after 40 DEG C of nitrogen dry up, with 100 μ L
After flowing phased soln, the analysis of 10 μ L sample introductions.
3. specificity：
The μ L of beagle dogs blank plasma 500 for being not given to Skullcapflavone-II are taken, add 4mL acetic acid second
Ester, 3500 × g centrifugation 10min after 3min are shaken, supernatant 3.5mL is taken after 40 DEG C of nitrogen dry up, flows phased soln with 100 μ L
Afterwards, 10 μ L sample introductions are analyzed.
Centrifuge tube number branch is taken, adds the μ L of Skullcapflavone-II standard liquids 10,40 DEG C of nitrogen dryings, addition does not have
Skullcapflavone-II beagle dogs blank plasma 500 μ L, vortex 30s mixing was given, adds the different Huang of new psoralea corylifolia
Ketone (internal standard, 100ng.mL-1) 10 μ L, 4mL ethyl acetate is added, 3500 × g centrifugation 10min after 3min is shaken, takes supernatant
3.5mL is after 40 DEG C of nitrogen dry up, after flowing phased soln with 100 μ L, the analysis of 10 μ L sample introductions.
As a result show, under the conditions of LC-MS/MS used by this experiment, plasma sample causes without miscellaneous peak to detected components
Interference, Skullcapflavone-II retention times exist in 3.09min or so, internal standard corylin retention time
2.88min left and right.Skullcapflavone-II and internal standard are not interfere with each other, and peak shape is good, and baseline is steady.
4. linear test
Beagle dog plasma standard curves：Centrifuge tube number branch is taken, precision adds various concentrations respectively
The μ L of Skullcapflavone-II standard liquids 10,40 DEG C of nitrogen dryings, addition were not given to Skullcapflavone-II
The μ L vortexs 30s of blank plasma 500 mix, it is respectively 0.001,0.005 to be made into the concentration containing Skullcapflavone-II,
0.01,0.05,0.1,0.5, Isosorbide-5-Nitrae μ g/mL plasma containing drug.Add neobavaisoflavone (internal standard, 100ngmL-1) 10 μ L,
4mL ethyl acetate is added, 3500 × g centrifugation 10min after 3min is shaken, takes supernatant 3.5mL to be used after 40 DEG C of nitrogen dry up
After 100 μ L flowing phased solns, the analysis of 10 μ L sample introductions.The ratio Y of Skullcapflavone-II and internal standard peak area is calculated, with peak
Area ratio Y makees to return to blood concentration X to be calculated, and obtains regression equation：Y=(0.00171 ± 0.00035) X+ (0.00067 ±
0.00029) (n=5), Skullcapflavone-II plasma concentrations are in 1~4000ngmL-1In the range of linear relationship it is good.
It is minimum to be quantitatively limited to 1ngmL-1。
5. the degree of accuracy and precision
It is 0.002 to prepare concentration containing Skullcapflavone-II according to beagle dog plasma standard curves method,
0.02and2 μ g/mL basic, normal, high plasma sample containing Skullcapflavone-II the, by " place of beagle dog plasma samples
Reason " processing method is handled respectively.Continuously do 3 days, each concentration respectively makees 5 parts of samples daily, calculates Skullcapflavone-II
Peak area As and internal standard peak area Ai ratio f, substitute into the plasma standard curve on the same day and try to achieve Skullcapflavone-II
Measured concentration, by measured concentration calculate in a few days, day to day precision and relative standard deviation (RSD), measured concentration is with adding concentration
Ratio be the degree of accuracy.As a result show, be in a few days respectively less than 10% with day to day precision RSD, the degree of accuracy meets the requirements.
6. measurement result
Above-mentioned 6 were only given Skullcapflavone-II contents in Skullcapflavone-II beagle dog plasmas
(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 as stationary phase, make by the mixed liquor of acetonitrile and water
For mobile phase, isocratic elution, Skullcapflavone-II retention times are 3.09min or so, internal standard neobavaisoflavone
Retention time is 2.88min or so, and 4.5min completes measure, and endogenous material does not disturb the measure of the two, while the side of pretreatment
Method is easy；Using organic solvent fluid page extraction, suitable for conventional determining；High sensitivity, blood plasma is minimum to be quantitatively limited to 1ng
mL-1, and the inventive method is quick, accurate, high sensitivity, easy to operate, the blood concentration for being Skullcapflavone-II is surveyed
It is fixed that foundation is provided, there is the prospect of new drug development.The plasma standard curve linear scope of this method is 1~4000ngmL-1, day
Interior and day to day precision RSD is respectively less than 10%.
Claims (6)
- A kind of 1. method for determining skullcapflavone II concentration in blood plasma, it is characterised in that plasma sample is preprocessed By its concentration of LC-MS system detectio, specific method comprises the following steps：(1) plasma sample pre-processes：Plasma sample is taken, internal standard neobavaisoflavone is added, adds a certain amount of ethyl acetate Organic solvent extraction is carried out, after taking supernatant nitrogen to dry up, pretreated plasma sample is obtained with flowing phased soln；(2) the pretreated plasma sample of step (1) is subjected to liquid chromatogram separation：In chromatographic condition, chromatographic column Agilent ZORBAX SB-C18Post；Mobile phase is the acetonitrile and water that volume ratio is 80: 20；Flow velocity is 0.2-0.5mLmin-1；Column temperature is 40℃；Type of elution is isocratic elution；(3) mass spectroscopy, condition are：Ion gun：Electro-spray ionization ionization source ESI；Ion detection mode：Multiple-reaction monitoring MRM；Ion polarity：Cation； Skullcapflavone-II collision voltages 23eV；Internal standard neobavaisoflavone collision voltage 29eV；Capillary voltage 4.5kV；Gas curtain gas Curtain gas：20psi；Ion gun Gas1：70psi；Ion gun Gas2：80psi；Desolventizing temperature： 400℃；CXP：9V；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：Using internal standard method, with skullcapflavone-II and internal standard neobavaisoflavone peak area ratio generation Enter the concentration that calibration curve equation calculates skullcapflavone-II.
- 2. the method for skullcapflavone-II concentration in measure blood plasma according to claim 1, it is characterised in that step Suddenly in (1), the μ L of plasma sample 500 are taken, add 100ngmL-1The μ L of internal standard neobavaisoflavone 20,4mL ethyl acetate is added, 3500 × g centrifuges 10min after shaking 3min, takes supernatant 3.5mL after 40 DEG C of nitrogen dry up, after flowing phased soln with 100 μ L, 10 μ L sample introductions are analyzed.
- 3. the method for skullcapflavone-II concentration in measure blood plasma according to claim 1, it is characterised in that step Suddenly in (2), chromatogram column length 150mm, internal diameter 2.1mm, packing material size are 5 μm.
- 4. the method for skullcapflavone-II concentration in measure blood plasma according to claim 1, it is characterised in that institute The blood plasma stated is the blood plasma for giving the medicine containing skullcapflavone-II.
- 5. the method for skullcapflavone-II concentration in measure blood plasma according to claim 1, it is characterised in that institute State the blood plasma that blood plasma is human or animal.
- 6. the method for skullcapflavone-II concentration in measure blood plasma according to claim 1, it is characterised in that institute It is dog plasma to state blood plasma.
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| KR101515067B1 (en) * | 2012-04-26 | 2015-04-27 | 한국생명공학연구원 | A pharmaceutical compositon containing SkullcapflavoneⅡ or pharmaceutically acceptable salt thereof for the prevention or treatment of asthma |
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