CN104101665B - Method for detecting chaetoglobosin concentration in blood plasma - Google Patents
Method for detecting chaetoglobosin concentration in blood plasma Download PDFInfo
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- CN104101665B CN104101665B CN201410380612.8A CN201410380612A CN104101665B CN 104101665 B CN104101665 B CN 104101665B CN 201410380612 A CN201410380612 A CN 201410380612A CN 104101665 B CN104101665 B CN 104101665B
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Abstract
The invention discloses a method for detecting chaetoglobosin concentration in blood plasma. The method adopts a liquid chromatography tandem mass spectrometry system to perform detection, a sample to be detected is firstly taken, a certain amount of organic menstruum protein precipitant is added to perform protein precipitation, and detection is performed through chromatographic column separation and a mass spectrum detector after pretreatment. The method is quick, accurate, high in sensitivity, simple and convenient to operate and suitable for concentration detection in the blood plasma.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of method measuring ball hair shell alkali first concentration in blood plasma.
Background technology
Ball hair shell alkali first (Chaetoglobins A) is from the endogenetic fungus chaetomium globosum (Chaetomiumglobosum) of medicinal plant cogongrass, be separated the alkaloids compounds obtained.Experiment in vitro proves that Chaetoglobins A has significant antitumor action, expression of proto-oncogenes and cancer cell diffusion can be suppressed, all having obvious suppression to MCF-7 cell strainHJ2mm and colon cancer cell line SW1116, is a kind of source new drugs compound with good DEVELOPMENT PROSPECT.Its structural formula is as follows:
At present not yet about the bibliographical information of Chaetoglobins A in vivoassay method.The present invention establishes the assay method of Chaetoglobins A in rat body, carries out pilot study by the drug metabolism situation of the method to Chaetoglobins A.
Summary of the invention
Technical matters to be solved by this invention sets up a kind of method that precise and high efficiency measures ball hair shell alkali first concentration in blood plasma.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
Measure a method for ball hair shell alkali first concentration in blood plasma, plasma sample is after pretreatment through its concentration of LC-MS systems axiol-ogy, and concrete grammar comprises the steps:
(1) plasma sample pre-service: get plasma sample, adds interior mark Tinidazole methanol solution and acetic acid aqueous solution, adds methyl alcohol or acetonitrile carries out albumen precipitation, gets supernatant after centrifugal;
(2) step (1) pretreated plasma sample is carried out liquid chromatography separation: in chromatographic condition, chromatographic column is Agilent ZORBAX SB-C18 post; Mobile phase: volume ratio is methyl alcohol and the water of 50:50; Flow velocity: 0.2-0.5mLmin
-1; Column temperature: 30-40 DEG C; Sample size 10 μ L; Type of elution is isocratic elution;
(3) mass spectroscopy, condition is: ion gun: electro-spray ionization ionization source ESI; Ion detection mode: multiple-reaction monitoring MRM; Ion polarity: positive ion; Capillary voltage: 3.0KV; Ion source temperature: 80-120 DEG C; Desolventizing temperature: 340-360 DEG C; Desolventizing gas: 400L/h; Taper hole voltage: 45V; Taper hole gas: 35L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion channel of detected object: ball hair shell alkali first, [M-H]+, m/z637.30 → 549.20, Dwell0.5, Cone45, Collision Energy35; Interior mark Tinidazole, [M-H]+, m/z248.20 → 121.00, Dwell0.5, Cone30, Collision Energy15;
(4) calculate: adopt internal standard method, substitute into the peak area ratio of ball hair shell alkali first and interior mark Tinidazole the concentration that typical curve equation calculates ball hair shell alkali first.
In step (1), get plasma sample 50 μ L, add interior mark 1000ngmL
-1tinidazole methanol solution 10 μ L and 20% (v/v) acetic acid aqueous solution 5 μ L, adds 190 μ L methyl alcohol or acetonitriles, and the centrifugal 10min of vibration 3min, 12000rpm, gets supernatant after centrifugal.
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 give the blood plasma containing ball hair shell alkali first medicine.
Wherein, described blood plasma is the blood plasma of human or animal.
Wherein, described blood plasma is rat plasma.
Beneficial effect: the inventive method compared with prior art has following advantage:
(1) preprocess method is easy: a step organic solvent precipitation of protein, is applicable to conventional determining.
(2) specificity is strong: adopt Agilent ZORBAX SB-C18 chromatographic column as Stationary liquid, the mixed liquor of first alcohol and water is as mobile phase, isocratic elution, Chaetoglobins A retention time is about 2.67min, interior mark Tinidazole retention time is about 2.43min, 4.5min completes mensuration, and endogenous material does not disturb the two mensuration.
(3) highly sensitive: blood plasma is minimum is quantitatively limited to 2ngmL
-1.
(4) the inventive method is quick, accurate, highly sensitive, easy and simple to handle, for the determination of plasma concentration of Chaetoglobins A provides foundation, has the prospect of new drug development.The plasma standard curve linear scope of this method is 2 ~ 1000ngmL
-1, be in a few days all less than 5% with day to day precision RSD.
Accompanying drawing explanation
Fig. 1 is the mass spectrogram that methyl alcohol adds Chaetoglobins A and interior mark reference substance.
Fig. 2 is the mass spectrogram of rat blank plasma.
Fig. 3 is the mass spectrogram that rat blank plasma adds Chaetoglobins A and interior mark reference substance.
Fig. 4 be after rat gives Chaetoglobins A plasma sample add again interior mark reference substance mass spectrogram.
Note: figure intermediate ion passage 1 is Chaetoglobins A, [M-H]+, m/z637.30 → 549.20, retention time is about 2.67min; Ion selector channel 2 is interior mark Tinidazole, [M-H]+, m/z248.20 → 121.00, retention time is about 2.43min.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment: the mensuration of Chaetoglobins A concentration in rat plasma
One, experiment material and instrument
Chaetoglobins A: be separated from cogongrass endogenetic fungus chaetomium globosum; Tinidazole (interior mark): be purchased from Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 100336-200402; Test water: ultrapure water; Methyl alcohol: chromatographically pure (Merck Company).
Quattro Micro LC/MS/MS LC-MS instrument (Waters, US, it is Waters2695 that efficient liquid phase instrument is joined by institute); Masslynx4.0 data handling system; The miniature vortex mixed instrument of WH-2 (Shanghai Hu Xi analytical instrument factory); AE240 electronic balance (Shanghai Mettler-Toledo, Inc.); Millipore Drict-Q5 water purification machine (French Millipore company); Biofuge PrimoR high-speed refrigerated centrifuge (German Heraeus company).
Two, liquid matter condition
1. liquid phase chromatogram condition
Chromatographic column: Agilent ZORBAX SB-C18 post (dp5 μm, 150mm ID × 2.1mm); Mobile phase: methanol-water (50:50, v/v); Flow velocity: 0.25mLmin
-1; Column temperature: 35 DEG C.
2. Mass Spectrometry Conditions
Ion gun: electro-spray ionization ionization source ESI; Ion detection mode: selectivity ion detection SIM; Ion polarity: positive ion; Capillary voltage: 3.0KV; Ion source temperature: 120 DEG C; Desolventizing temperature: 350 DEG C; Desolventizing gas: 400L/h; Taper hole voltage: 45V; Taper hole gas: 35L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion channel of detected object: Chaetoglobins A, [M-H]+, m/z637.30 → 549.20, Dwell0.5, Cone45, Collision Energy35; Interior mark Tinidazole, [M-H]+, m/z248.20 → 121.00, Dwell0.5, Cone30, Collision Energy15.
Three, experimentation:
The preparation of 1.Chaetoglobins A standard solution:
Precision takes Chaetoglobins A10.72mg, is placed in 10mL volumetric flask, adds methyl alcohol and dissolves and be settled to scale, shake up, obtain 1.072mgmL
-1the storing solution of Chaetoglobins A.Precision measures appropriate storing solution and dilutes successively with methyl alcohol, be made into concentration be respectively 10,25,50,100,250,500,1000,2500,5000ngmL
-1chaetoglobins A standard solution.
2. the Acquire and process of rat plasma sample:
Rat tail vein injection Chaetoglobins solution A, dosage is 1mgkg
-1, before injection and after injection 5,10,20,30,45,60,80,100min, 2,2.5,3,4h eye socket takes a blood sample 200 μ L, in injection calparine pipe, the centrifugal 10min of 3000rpm, prepares blood plasma.Get rat plasma 50 μ L, add 1000ngmL
-1interior mark Tinidazole methanol solution 10 μ L, 20% (v/v) acetic acid aqueous solution 5 μ L, methyl alcohol 190 μ L, vibration 3min carries out albumen precipitation, the centrifugal 10min of 12000rpm, and supernatant 10 μ L sample introduction is analyzed.
3. specificity:
Get the rat blank plasma 50 μ L not giving Chaetoglobins A, add 20% (v/v) acetic acid aqueous solution 5 μ L, 200 μ L methyl alcohol, vibration 3min carries out albumen precipitation, the centrifugal 10min of 12000rpm, and supernatant 10 μ L sample introduction is analyzed.
Get 1.5mL plastic centrifuge tube number to prop up, add Chaetoglobins A standard solution 10 μ L, 40 DEG C of nitrogen dry up, add the rat blank plasma 50 μ L not giving Chaetoglobins A, vortex 30s mixes, and adds Tinidazole methanol solution (interior mark, 1000ngmL
-1) 10 μ L, 20% (v/v) acetic acid aqueous solution 5 μ L, methyl alcohol 190 μ L, vibration 3min carries out albumen precipitation, the centrifugal 10min of 12000rpm, and supernatant 10 μ L sample introduction is analyzed.
Result shows, and under the LC-MS-MS condition that this experiment adopts, plasma sample causes interference without assorted peak to detected components, and Chaetoglobins A retention time is at about 2.67min, and interior mark Tinidazole retention time is at about 2.43min.Chaetoglobins A and interior mark do not interfere with each other, and peak shape is good, and baseline is steady.
4. linear test
Rat plasma typical curve: get 1.5mL plastic centrifuge tube number and prop up, the accurate Chaetoglobins A standard solution 10 μ L adding variable concentrations respectively, 40 DEG C of nitrogen dry up, add the rat blank plasma 50 μ L vortex 30s not giving Chaetoglobins A to mix, the concentration be made into containing Chaetoglobins A be respectively 2,5,10,20,50,100,200,500,1000ngmL
-1plasma containing drug.Add Tinidazole methanol solution (interior mark, 1000ngmL
-1) 10 μ L, 20% (v/v) acetic acid aqueous solution 5 μ L, methyl alcohol 190 μ L, vibration 3min carries out albumen precipitation, the centrifugal 10min of 12000rpm, and supernatant 10 μ L sample introduction is analyzed.Calculate the ratio f of Chaetoglobins A and interior mark peak area, do to return calculating to blood concentration C with peak area ratio f, obtain regression equation: f=0.0034 × C+0.0009 (r=0.999), W=1/cc, Chaetoglobins A plasma concentration at 2 ~ 1000ngmL
-1scope internal linear relation is good.Minimumly quantitatively be limited to 2ngmL
-1.
5. accuracy and precision
According to the preparation of rat plasma typical curve method containing Chaetoglobins A concentration be 5,50,500ngmL
-1basic, normal, high containing Chaetoglobins A plasma sample, process respectively by " process of rat plasma sample " disposal route.Do 3 days continuously, every day, each concentration respectively did 5 increment product, calculate the ratio f of Chaetoglobins A peak area As and interior mark peak area Ai, substitute in the plasma standard curve on the same day and try to achieve Chaetoglobins A measured concentration, calculated in a few days by measured concentration, day to day precision and relative standard deviation (RSD), measured concentration is accuracy with the ratio adding concentration.Result shows, and be in a few days all less than 10% with day to day precision RSD, accuracy meets the requirements.
6. measurement result
Above-mentioned 6 gave Chaetoglobins A content (after administration 30min) in the rat plasma of Chaetoglobins A be respectively 45.62,56.55,65.54,87.59,48.21,55.60ngmL
-1.
Claims (6)
1. measure a method for ball hair shell alkali first Chaetoglobins A concentration in blood plasma, it is characterized in that, plasma sample is after pretreatment through its concentration of LC-MS systems axiol-ogy, and concrete grammar comprises the steps:
(1) plasma sample pre-service: get plasma sample, adds interior mark Tinidazole methanol solution and acetic acid aqueous solution, adds methyl alcohol or acetonitrile carries out albumen precipitation, gets supernatant after centrifugal;
(2) step (1) pretreated plasma sample is carried out liquid chromatography separation: in chromatographic condition, chromatographic column is Agilent ZORBAX SB-C18 post; Mobile phase: volume ratio is methyl alcohol and the water of 50:50; Flow velocity: 0.2-0.5mLmin
-1; Column temperature: 30-40 DEG C; Sample size 10 μ L; Type of elution is isocratic elution;
(3) mass spectroscopy, condition is: ion gun: electro-spray ionization ionization source ESI; Ion detection mode: multiple-reaction monitoring MRM; Ion polarity: positive ion; Capillary voltage: 3.0KV; Ion source temperature: 80-120 DEG C; Desolventizing temperature: 340-360 DEG C; Desolventizing gas: 400L/h; Taper hole voltage: 45V; Taper hole gas: 35L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion channel of detected object: ball hair shell alkali first Chaetoglobins A, [M-H]+, m/z 637.30 → 549.20, Dwell 0.5, Cone 45, Collision Energy 35; Interior mark Tinidazole, [M-H]+, m/z 248.20 → 121.00, Dwell 0.5, Cone 30, Collision Energy 15;
(4) calculate: adopt internal standard method, substitute into the peak area ratio of ball hair shell alkali first Chaetoglobins A and interior mark Tinidazole the concentration that typical curve equation calculates ball hair shell alkali first Chaetoglobins A.
2. the method for ball hair shell alkali first Chaetoglobins A concentration in mensuration blood plasma according to claim 1, is characterized in that, in step (1), get plasma sample 50 μ L, add interior mark 1000ngmL
-1tinidazole methanol solution 10 μ L and 20v/v% acetic acid aqueous solution 5 μ L, adds 190 μ L methyl alcohol or acetonitriles, and the centrifugal 10min of vibration 3min, 12000rpm, gets supernatant after centrifugal.
3. the method for ball hair shell alkali first Chaetoglobins A concentration in mensuration blood plasma according to claim 1, it is characterized in that, in step (2), chromatogram column length is 150mm, and internal diameter is 2.1mm, and packing material size is 5 μm.
4. the method for ball hair shell alkali first Chaetoglobins A concentration in mensuration blood plasma according to claim 1, it is characterized in that, described blood plasma is give the blood plasma containing ball hair shell alkali first Chaetoglobins A medicine.
5. the method for ball hair shell alkali first Chaetoglobins A concentration in mensuration blood plasma according to claim 1, it is characterized in that, described blood plasma is the blood plasma of human or animal.
6. the method for ball hair shell alkali first Chaetoglobins A concentration in mensuration blood plasma according to claim 1, it is characterized in that, described blood plasma is rat plasma.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103694247A (en) * | 2013-12-06 | 2014-04-02 | 秦建春 | Compound Chaetomugilide A and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103694247A (en) * | 2013-12-06 | 2014-04-02 | 秦建春 | Compound Chaetomugilide A and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Chaetoglobins A and B, two unusual alkaloids from endophytic Chaetomium globosum culture;Hui Ming Ge等;《Chem. Commun.》;20081231;第5978-5980页 * |
| Two new azaphilone alkaloids dimers from endophyte Chaetomium fusiforme of the liverwort Scapania verrucosa Heeg.;Wei Peng等;《Biochemical Systematics and Ecology》;20121231;第45卷;第124-126页 * |
| 内生真菌代谢产物球毛壳菌素A的液相色谱分析;程敬丽等;《农药》;20110131;第50卷(第1期);第43-45页 * |
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