CN106153766B - A kind of method of 8- table diosbulbin E Acetate concentrations in measurement blood plasma - Google Patents
A kind of method of 8- table diosbulbin E Acetate concentrations in measurement blood plasma Download PDFInfo
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- CN106153766B CN106153766B CN201610460967.7A CN201610460967A CN106153766B CN 106153766 B CN106153766 B CN 106153766B CN 201610460967 A CN201610460967 A CN 201610460967A CN 106153766 B CN106153766 B CN 106153766B
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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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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
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Abstract
The invention discloses a kind of methods of 8 table diosbulbin E Acetate concentrations in measurement blood plasma, using LC-MS system measurement, first take sample to be tested, a certain amount of organic solvent protein precipitant is added and carries out albumen precipitation, after pretreatment, through chromatography post separation, detected with mass detector, the method of the present invention quickly, it is accurate, high sensitivity, easy to operate, be suitable for measuring the concentration of 8 table diosbulbin E acetic acid esters in blood plasma.
Description
Technical field
The invention belongs to technical field of biological, and in particular to 8- table diosbulbin E acetic acid esters is dense in a kind of measurement blood plasma
The method of degree.
Background technology
8- table diosbulbin E acetic acid esters (8-epidiosbulbin E acetate) is from Dioscoreaceae Dioscorea perennial grass
The isolated positive Diterpenes noval chemical compound of furans in the dry tuber of this plant air potato (Dioscorea bulbifera L.).
Experiment in vitro proves that 8- table diosbulbin E acetic acid esters has the bioactivity such as significantly antitumor, antimycotic, anti-inflammatory, is a kind of tool
There is the new drug source compound of good development prospect.Its structural formula is as follows：
Document report not yet about 8- table diosbulbin E acetic acid esters in vivoassay methods at present.The present invention establishes 8- tables
Assay method of the diosbulbin E acetic acid esters in rat body, by this method to the drug metabolism situation of 8- table diosbulbin E acetic acid esters
Carry out pilot study.
Invention content
The technical problem to be solved by the present invention is to establish a kind of precise and high efficiency to measure 8- table diosbulbin E acetic acid esters in blood plasma
The method of 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 8- table diosbulbin E Acetate concentrations in measurement blood plasma, joins after plasma sample is preprocessed through liquid matter
With its concentration of system detectio, specific method includes the following steps：
(1) plasma sample pre-processes：Take plasma sample, internal standard Tinidazole methanol solution be added, be added methanol or acetonitrile into
Row albumen precipitation takes supernatant after centrifugation；
(2) the pretreated plasma sample of step (1) is subjected to liquid chromatogram separation：Chromatographic column is in chromatographic condition
Phenomenex Gemini C18 columns；Mobile phase：The methanol and 0.1% aqueous formic acid that volume ratio is 70: 30；Flow velocity：0.2-
0.5mL·min-1；Column temperature：30-40℃；5 μ L of sample size；Type of elution is isocratic elution；
(3) mass spectroscopy, condition are：Ion source：Electro-spray ionization ionization source ESI；Ion detection mode：More reaction prisons
Survey MRM；Ion polarity：Cation；Capillary voltage：4000V；Dry temperature degree：400℃；Desolventizing gas and taper hole gas are
High pure nitrogen；Detect the ion channel of object：8- table diosbulbin E acetic acid esters, [M-Na]+, m/z 411.1 → 351.0, Dwell
100, Fragmentor 150, Collision Energy 25；Internal standard Tinidazole, [M-H]+, m/z 248.2 → 121.0,
Dwell 100, Fragmentor 110, Collision Energy 10；
(4) it calculates：Using internal standard method, is substituted into and marked with the peak area ratio of 8- tables diosbulbin E acetic acid esters and internal standard Tinidazole
The concentration of directrix curve equation calculation 8- table diosbulbin E acetic acid esters.
In step (1), 50 μ L of plasma sample are taken, internal standard 800ngmL is added-1Tinidazole methanol solution 10 μ L and 190 μ L
Methanol or acetonitrile, vibrate 3min, and 12000rpm centrifuges 10min, supernatant is taken after centrifugation.
In step (2), chromatogram column length 100mm, internal diameter 2.0mm, packing material size are 3 μm.
Wherein, the blood plasma is the blood plasma for giving the diosbulbin E acetic acid esters drugs of table containing 8-.
Wherein, the blood plasma is the blood plasma of human or animal.
Wherein, the blood plasma is rat plasma.
Advantageous effect：The method of the present invention has following advantage compared with prior art：
(1) preprocess method is easy：One step organic solvent precipitation of protein is suitable for conventional determining.
(2) specificity is strong：Using Phenomenex Gemini C18 chromatographic columns as stationary phase, the mixing of first alcohol and water
Liquid is as mobile phase, and isocratic elution, 8- table diosbulbin E acetic acid esters retention times are 1.5min or so, when internal standard Tinidazole retains
Between for 1.3min or so, 2min completes to measure, the measurement that both endogenous material is not interfered.
(3) high sensitivity：Blood plasma is minimum to be quantitatively limited to 1ngmL-1。
(4) the method for the present invention quickly, it is accurate, high sensitivity, easy to operate, be that the blood medicine of 8- table diosbulbin E acetic acid esters is dense
Degree, which measures, provides foundation, the foreground with new drug development.The plasma standard curve linear ranging from 1~1000ng of this method
mL-1, in a few days 8.4% is respectively less than with day to day precision RSD.
Description of the drawings
Fig. 1 is the mass spectrogram that 8- table diosbulbin E acetic acid esters and internal standard reference substance is added in methanol.
Fig. 2 is the mass spectrogram of rat blank plasma.
Fig. 3 is the mass spectrogram that 8- table diosbulbin E acetic acid esters and internal standard reference substance is added in rat blank plasma.
Fig. 4 is the mass spectrogram that rat gives that plasma sample after 8- table diosbulbin E acetic acid esters adds internal standard reference substance.
Note：Fig. 1~Fig. 4 intermediate ions channel 1 be 8- table diosbulbin E acetic acid esters, [M-Na]+, m/z 411.1 → 351.0, protect
It is 1.5min or so to stay the time；Ion selector channel 2 be internal standard Tinidazole, [M-H]+, m/z 248.2 → 121.0, retention time
For 1.3min or so.
Specific implementation mode
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.
Embodiment：The measurement of 8- tables diosbulbin E Acetate concentrations in rat plasma
One, experiment material and instrument
8- table diosbulbin E acetic acid esters：It is isolated from the dry tuber of air potato；Tinidazole (internal standard)：It is purchased from Chinese drug biology
Product examines and determine institute, lot number：100336-200703；Test water：Ultra-pure water；Methanol：Chromatographically pure (Merck Company).
(Agilent companies of the U.S., matched efficient liquid phase instrument are Agilent G6430LC-MS/MS LC-MS instrument
Agilent 1290)；MassHunter data processing systems (version is B.05.00)；The miniature vortex mixed instruments of WH-2 (Shanghai
Hu Xi analytical instrument factory)；AE240 electronic balances (Shanghai Mettler-Toledo, Inc.)；Millipore Drict-Q5 are pure
Water dispenser (French Millipore companies)；Biofuge PrimoR high-speed refrigerated centrifuges (German Heraeus companies).
Two, liquid matter condition
1. liquid phase chromatogram condition
Chromatographic column：Phenomenex Gemini C18 columns (3 μm of dp, 100mm ID × 2.0mm)；Mobile phase：Methanol-
0.1% aqueous formic acid (70: 30, v/v)；Flow velocity：0.2mL·min-1；Column temperature：35℃.
2. Mass Spectrometry Conditions
Ion source：Electro-spray ionization ionization source ESI；Ion detection mode：Multiple-reaction monitoring MRM；Ion polarity：Just from
Son；Capillary voltage：4000V；Dry temperature degree：400℃；Desolventizing gas and taper hole gas are high pure nitrogen；Detect object
Ion channel：8- table diosbulbin E acetic acid esters, [M-Na]+, m/z 411.1 → 351.0, Dwell 100, Fragmentor 150,
Collision Energy 25；Internal standard Tinidazole, [M-H]+, m/z 248.2 → 121.0, Dwell 100, Fragmentor
110, Collision Energy 10.
Three, experimentation：
The preparation of 1.8- table diosbulbin E acetate standard solution：
Precision weighs 8- table diosbulbin E acetic acid esters 10.14mg, is placed in 10mL volumetric flasks, and methanol dissolving and constant volume is added
To scale, shake up to get 1.014mgmL-1The storing solution of 8- table diosbulbin E acetic acid esters.Precision measures appropriate storing solution with first
Alcohol dilutes successively, be made into concentration be respectively 5,25,50,100,250,500,1000,2500,5000ngmL-18- table air potatoes
Plain E acetate standards solution.
2. the acquisition and processing of rat plasma sample：
Rat tail vein injects 8- table diosbulbin E acetate solutions, dosage 1mgkg-1, before injection and after injection
5,10,20,30,45min, 1,2,2.5,3,4h eye sockets are taken a blood sample 200 μ L, are injected in calparine pipe, 3000rpm centrifuges 10min, prepares
Blood plasma.50 μ L of rat plasma are taken, 800ngmL is added-110 μ L of internal standard Tinidazole methanol solution, 190 μ L of methanol, oscillation 3min into
Row albumen precipitation, 12000rpm centrifuge 10min, and sample introduction is analyzed by 5 μ L of supernatant.
3. specificity：
The 50 μ L of rat blank plasma for being not given to 8- table diosbulbin E acetic acid esters are taken, 200 μ L methanol, oscillation is added
3min carries out albumen precipitation, and 12000rpm centrifuges 10min, and sample introduction is analyzed by 5 μ L of supernatant.
1.5mL plastic centrifuge tube number branch is taken, 10 μ L of 8- table diosbulbin E acetate standards solution are added, 40 DEG C of nitrogen dry up,
The 50 μ L of rat blank plasma for being not given to 8- table diosbulbin E acetic acid esters are added, Tinidazole methanol is added in vortex 30s mixings
Solution (internal standard, 800ngmL-1) 10 μ L, 190 μ L of methanol, oscillation 3min progress albumen precipitations, 12000rpm centrifugation 10min, on
Sample introduction is analyzed by 5 μ L of clear liquid.
The results show that under the conditions of LC-MS/MS used by this experiment, plasma sample causes detected components without miscellaneous peak
Interference, 8- table diosbulbin E acetic acid esters retention times are in 1.5min or so, and internal standard Tinidazole retention time is in 1.3min or so.8-
Table diosbulbin E acetic acid esters and internal standard are not interfere with each other, and peak shape is good, and baseline is steady.
4. linear test
Rat plasma standard curve：1.5mL plastic centrifuge tube number branch is taken, respectively the accurate 8- table air potatoes that various concentration is added
The rat blank for being not given to 8- table diosbulbin E acetic acid esters is added in 10 μ L of plain E acetate standards solution, 40 DEG C of nitrogen dryings
50 μ L vortex 30s mixings of blood plasma, the concentration for being made into the diosbulbin E acetic acid esters of table containing 8- is respectively 1,5,10,20,50,100,200,
500、1000ng·mL-1Plasma containing drug.Tinidazole methanol solution (internal standard, 800ngmL is added-1) 10 μ L, 190 μ L of methanol,
It vibrates 3min and carries out albumen precipitation, 12000rpm centrifuges 10min, and sample introduction is analyzed by 5 μ L of supernatant.Calculate 8- table diosbulbin E acetic acid
The ratio f of ester and internal standard peak area makees blood concentration C with peak area ratio f to return calculating, obtains regression equation：F=0.0025
× C-0.0012 (r=0.997), W=1/cc, 8- table diosbulbin E acetic acid esters plasma concentrations are in 1~1000ngmL-1In range
Linear relationship is good.It is minimum to be quantitatively limited to 1ngmL-1。
5. accuracy and precision
According to rat plasma standard curve method prepare the diosbulbin E Acetate concentrations of table containing 8- be 2,50,500ngmL-1
The basic, normal, high diosbulbin E acetic acid esters plasma samples of table containing 8-, locate respectively by " processing of rat plasma sample " processing method
Reason.It continuously does 3 days, each concentration respectively makees 5 parts of samples daily, calculates 8- tables diosbulbin E acetic acid esters peak area As and internal standard peak face
The ratio f of product Ai, substitutes into the plasma standard curve on the same day and acquires 8- table diosbulbin E acetic acid esters measured concentrations, by measured concentration
Calculate in a few days, day to day precision and relative standard deviation (RSD), measured concentration and the ratio that concentration is added are accuracy.Knot
Fruit shows, is in a few days respectively less than 8.4% with day to day precision RSD, accuracy meets the requirements.
6. measurement result
Above-mentioned 6 were only given the (administration of 8- table diosbulbin E acetate contents in the rat plasma of 8- table diosbulbin E acetic acid esters
30min afterwards) be respectively 35.58,40.27,78.37,48.19,43.38,50.33ngmL-1。
Claims (6)
1. a kind of method measuring 8- table diosbulbin E Acetate concentrations in blood plasma, which is characterized in that plasma sample is after pretreatment
Through its concentration of LC-MS system detectio, specific method includes the following steps：
（1）Plasma sample pre-processes：Plasma sample is taken, internal standard Tinidazole methanol solution is added, methanol is added or acetonitrile carries out egg
White precipitation, supernatant is taken after centrifugation；
（2）By step（1）Pretreated plasma sample carries out liquid chromatogram separation：Chromatographic column is in chromatographic condition
Phenomenex Gemini C18 columns；Mobile phase：Volume ratio is 70:30 methanol and 0.1% aqueous formic acid；Flow velocity：0.2-
0.5 mL·min-1；Column temperature：30-40 ℃；Sample size：5 μL；Type of elution is isocratic elution；
（3）Mass spectroscopy, condition are：Ion source：Electro-spray ionization ionization source ESI；Ion detection mode：Multiple-reaction monitoring
MRM；Ion polarity：Cation；Capillary voltage：4000 V；Dry temperature degree：400 ℃；Desolventizing gas and taper hole gas are
High pure nitrogen；Detect the ion channel of object：8- table diosbulbin E acetic acid esters, [M-Na]+, m/z 411.1 → 351.0, when being resident
Between 100, fragmentation voltage 150, collision energy 25；Internal standard Tinidazole, [M-H]+, m/z 248.2 → 121.0, residence time
100, fragmentation voltage 110, collision energy 10；
（4）It calculates：Using internal standard method, it is bent that standard is substituted into the peak area ratio of 8- tables diosbulbin E acetic acid esters and internal standard Tinidazole
The concentration of line equation calculation 8- table diosbulbin E acetic acid esters.
2. the method according to claim 1 for measuring 8- table diosbulbin E Acetate concentrations in blood plasma, which is characterized in that step
Suddenly（1）In, 50 μ L of plasma sample are taken, 800 ngmL of internal standard is added-110 μ L of Tinidazole methanol solution and 190 μ L methanol or
Acetonitrile, vibrates 3 min, and 12000 rpm centrifuge 10 min, supernatant is taken after centrifugation.
3. the method according to claim 1 for measuring 8- table diosbulbin E Acetate concentrations in blood plasma, which is characterized in that step
Suddenly（2）In, chromatogram column length is 100 mm, and internal diameter is 2.0 mm, and packing material size is 3 μm.
4. the method according to claim 1 for measuring 8- table diosbulbin E Acetate concentrations in blood plasma, which is characterized in that institute
The blood plasma stated is the blood plasma for giving the diosbulbin E acetic acid esters drugs of table containing 8-.
5. the method according to claim 1 for measuring 8- table diosbulbin E Acetate concentrations in blood plasma, which is characterized in that institute
State the blood plasma that blood plasma is human or animal.
6. the method according to claim 1 for measuring 8- table diosbulbin E Acetate concentrations in blood plasma, which is characterized in that institute
It is rat plasma to state blood plasma.
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| CN110568097B (en) * | 2019-09-10 | 2022-06-24 | 张家港市中医医院 | Method for measuring concentration of glycyrrhiza isoflavone B in blood plasma |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102070646A (en) * | 2011-01-11 | 2011-05-25 | 山东省中医药研究院 | Extraction method of Diosbulbin B |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102070646A (en) * | 2011-01-11 | 2011-05-25 | 山东省中医药研究院 | Extraction method of Diosbulbin B |
Non-Patent Citations (3)
| Title |
|---|
| Dongju Lin et al..In Vitro and in Vivo Studies of the Metabolic Activation of 8‑Epidiosbulbin E Acetate.《Chem. Res. Toxicol.》.2015,第28卷 * |
| Dongju Lin et al..Role of Metabolic Activation in 8‑Epidiosbulbin E Acetate-Induced Liver Injury: Mechanism of Action of the Hepatotoxic Furanoid.《Chem. Res. Toxicol.》.2016,第29卷 * |
| 黄药子二萜内酯类成分化学、药理及毒性研究进展;王君明 等;《中医学报》;20111101;第26卷(第11期);第1319-1321页 * |
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