CN107014915A - The quantitative detecting method of abiraterone in whole blood - Google Patents
The quantitative detecting method of abiraterone in whole blood Download PDFInfo
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- abiraterone
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- 239000008280 blood Substances 0.000 title claims abstract description 83
- 229960000853 abiraterone Drugs 0.000 title claims abstract description 80
- GZOSMCIZMLWJML-VJLLXTKPSA-N abiraterone Chemical compound C([C@H]1[C@H]2[C@@H]([C@]3(CC[C@H](O)CC3=CC2)C)CC[C@@]11C)C=C1C1=CC=CN=C1 GZOSMCIZMLWJML-VJLLXTKPSA-N 0.000 title claims abstract description 80
- 238000000034 method Methods 0.000 title claims abstract description 20
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 46
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- HMUNWXXNJPVALC-UHFFFAOYSA-N 1-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C(CN1CC2=C(CC1)NN=N2)=O HMUNWXXNJPVALC-UHFFFAOYSA-N 0.000 description 4
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Classifications
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N2030/062—Preparation extracting sample from raw material
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- Physics & Mathematics (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of quantitative detecting method of abiraterone in whole blood, using whole blood as object to be measured, follow the steps below successively：Centrifuge, centrifuged after being redissolved after the drying of ethyl acetate layer nitrogen stream with 20% acetonitrile solution, Aspirate supernatant I after ethyl acetate vibration whirlpool is added in whole blood lysate；Alkaline diatomite is weighed to be put into glass chromatography column, vibration processing, then supernatant I is added to be eluted with pure water, 20% methanol aqueous solution, chromatogram methanol successively, centrifuged after being redissolved after the drying of eluent nitrogen stream with 20% acetonitrile solution, Aspirate supernatant II carries out highly effective liquid phase chromatographic system analysis, abiraterone medicine peak area Y is obtained, it is the abiraterone concentration of supernatant II to substitute into formula Y=27524X+1956.6, X；Then through conversion, the abiraterone concentration in whole blood to be measured is obtained.
Description
Technical field
The present invention relates to the quantitative detecting method of abiraterone in a kind of animal (including people) whole blood.
Background technology
Prostate cancer is a kind of common malignant tumour of American-European male, and newly-increased diagnosis number is about 240000 every year, is swollen
The tumour of knurl deaths in men rate the 2nd, and metastatic is strong.According to statistics, about 28000 people in 2012 die from the disease.Although before China
The row gland cancer incidence of disease is far below western countries, but with living-pattern preservation, the popularization of prostate cancer specific antigen examination,
The number of patients of China's prostate cancer also linear type ascendant trend, and wherein most of develop into advanced prostate cancer.It is sent out
The main mechanism of exhibition is lasting stimulation of the intracellular androgen receptor by male sex hormone, at present for the first choice of prostate cancer
Treatment method is castration, but research display initial stage it is effective after after averagely 36 months a big chunk patient evolution supported for castration
Refractory prostate cancer, finds that patient may continue to synthesize a small amount of male after treatment is received in prostate gland cancer cell after research
Hormone.Subsequent research discovery, first generation androgen receptor antagonists ketoconazole controllable male sex hormone axle, but therapeutic effect
It is of short duration, and ketoconazole is very poor for the tolerance for suppressing male sex hormone synthesis.Multiple studies have shown that, reached in serum testosterone
Androgen receptor signal shaft in gesture level, prostate gland cancer cell can still keep activity, and it is bred with important
Influence.The increase of serum prostate cancer specific antigen can be by two wires hormone therapy medicine after clinical research confirmation, castration
Thing is reduced, and androgen receptor pathway activity is unrelated with testosterone cyclical level.
Abiraterone is a kind of antiandrogen medicine, is the CYP17 enzyme inhibitors during male hormone metabolism, belongs to interior point
Treatment category is secreted, the medicine is by blocking the key enzyme of androgen biosynthesis to reduce testosterone levels, except testis, adrenal gland come
Outside the testosterone in source, the androgen synthesis inside tumor of prostate in microenvironment can also be reduced.Abiraterone can be with strong inhibition
17 α-hydroxylase/C17,2- lyases (CYP17), so that the biosynthesis of androgen is significantly inhibited, in the drug metabolism of mouse
In kinetic model abiraterone for CYP17 inhibitory activity considerably beyond ketoconazole.Phase I clinical trial result shows, Ah
Bit dragon can not only suppress testosterone levels well under castration environment, and with good tolerance, also not show
The toxic reaction gone out as ketoconazole.The clinical test of open, single centre and dosage escalation shows lasting abiraterone treatment
Can reduce prostate cancer specific antigen in 50%~57% patients serum, and II phases clinic recommendation therapeutic dose is
1000mg/d, can suppress the synthesis of glucocorticoid and androgen simultaneously under this dosage.Abiraterone chemical structural formula such as formula 1
It is shown.
At present, the quantitative detection and analysis on abiraterone, there is not yet Related domestic documents are reported.Southwest Jiaotong University
Pharmaceutical engineering institute Wu pine etc. has carried out the synthesis and its quality testing research of abiraterone acetate ester, and it uses Luna C18
Chromatographic column (250mm × 4.6mm, 5 μm), flow velocity 1.0ml/min, Detection wavelength 220nm, 40 DEG C of column temperature, mobile phase A is mutually first
Alcohol-acetonitrile (80%-20%), Mobile phase B are mutually water, gradient elution (elution program is shown in Table 1), quantitative analysis acetic acid Ah's bit
The content of imperial ester and the abiraterone existed as impurity.Testing result shows that abiraterone is separated preferably with other impurities, peak
Shape is also very symmetrical, unmatched exhibition or conditions of streaking.But the research is the detection carried out in synthetic reaction extract, and sample
Early stage purified treatment has been carried out in product, is disturbed in response sample without the intracellular biological such as albumen, lipid components impurity.
The gradient elution program of table 1.
Foreign periodical reports India Kumar etc. and uses abiraterone in RP-HPLC method quantitative analyses rat plasma.
Document display uses Betasil C18 chromatographic columns, and profit is extracted with ethyl acetate, and rat plasma is quantitatively have detected under room temperature environment
Middle abiraterone content.Its rate of recovery is more than 70%, and mobile phase is acetonitrile-water -0.01M potassium dihydrogen phosphates (pH3.0)=55-
5-40(V/V/V)。
It can be seen from document, abiraterone HPLC detections being capable of abiraterone in quantitative analysis biological sample in animal blood plasma
Content, as a result quantitative accurate, sensitive, reproducible, in a few days and in the daytime the coefficient of variation is smaller, disclosure satisfy that Ah's bit in animal body
Imperial Pharmacokinetics research.But abiraterone content, animal blood plasma in the research animal blood plasma that has been quantitative analysis
To remove a class biological sample of the cell components such as red blood cell, leucocyte, lymphocyte, intracellular substantial amounts of biological impurities are
It is eliminated, so that plasma sample is compared to relatively cleaner.Therefore, current research is that quantitatively have detected blood plasma or blood
Abiraterone content in clear, not yet learns Ah's bit in all kinds of haemocytes contained in blood (including red blood cell, leucocyte etc.)
Imperial content, it is also difficult to obtain abiraterone content in whole blood.Haemocyte as medicine important a bank and buffer, sometimes
Time needs distribution characteristics of the thoroughly evaluating medicine in haemocyte, so that ensure synergistic activity in terms of drug combination, reduction connection
Share the side effect of medicine.Therefore, know that abiraterone content has its specific function in whole blood.
The content of the invention
The technical problem to be solved in the present invention is to provide abiraterone content in a kind of whole blood (including blood plasma and haemocyte)
Accurate, quantitative detecting method.
In order to solve the above-mentioned technical problem, the present invention provides a kind of quantitative detecting method of abiraterone in whole blood, with complete
Blood is followed the steps below successively as object to be measured：
1) whole blood lysate 0.4mL, is drawn in lidded container (centrifuge tube with cover), adds ethyl acetate 3mL, oscillator
Whirlpool 2 ± 0.2min, 8000 ± 500r/min 5 ± 0.5min of high speed centrifugation, must be located at the ethyl acetate layer on upper strata, be located at respectively
The residue of lower floor；
Added in residue 2mL ethyl acetate repeat above-mentioned oscillator whirlpool, high speed centrifugation (oscillator whirlpool 2 ±
5 ± 0.5min of 0.2min, 8000 ± 500r/min high speed centrifugation)；That is, residue repeats extraction once；
Merge the ethyl acetate layer twice obtained by centrifugation；Nitrogen stream is dried up in 45 ± 1 DEG C of water-baths, and the dried object of gained is used
0.2mL 20% (volume %) acetonitrile solution redissolves, and whirlpool mixes 1 ± 0.2min, 18000 ± 500r/min high speed centrifugations 5
After ± 0.5min, Aspirate supernatant I；
Remarks explanation：The volume of this supernatant I is 0.2mL；That is, the precipitation that high speed centrifugation is produced is very little, can be neglected
Disregard；
2) alkaline diatomite (pH is 8.5 diatomite) 4g, is weighed to be put into glass chromatography column (10 × 200mm specifications),
Vibration handles 5 ± 0.5min (in 100Hz oscillating column pumps)；
3) supernatant I (0.2mL) in and then by step 1) all adds steps 2) glass chromatography column in, first use 5mL
Pure water is eluted, and is then eluted with 20% methanol (v/v) aqueous solution 5mL；Finally eluted with 5mL chromatograms methanol, collect chromatogram methanol
Corresponding whole eluents, nitrogen stream is dried up in 45 ± 1 DEG C of water-baths, and dried object is answered with 0.2mL 20% acetonitrile solution
Molten, whirlpool is mixed after 1 ± 0.2min, 18000 ± 500r/min high speed centrifugations, 5 ± 0.5min, and Aspirate supernatant II is carried out subsequently
Highly effective liquid phase chromatographic system analysis；
Remarks explanation：The volume of this supernatant II is 0.2mL；That is, the precipitation that high speed centrifugation is produced is very little, can be neglected
Disregard；The μ L of sample size 20 of following step, i.e. carry out subsequent step equivalent to the supernatant II of 1/10 volume has been taken；
4), Japanese Shimadzu shimadzu 20AT highly effective liquid phase chromatographic systems, Zhejiang University's intelligence reaches N2000 chromatographic work stations,
Chromatographic column uses Thermo Hypersil BDS C18 chromatographic columns (3 μm, 250mm × 4.6mm)；Mobile phase be acetonitrile-water-
0.01M sodium dihydrogen phosphates (pH 2.5)=60:5:35 (v/v), flow velocity 1.0mL/min, 40 DEG C of column temperature, ultraviolet detection wavelength
254nm, the μ L of sample size 20；
5), using step 4) obtained by abiraterone medicine peak area as Y, substitute into formula Y=27524X+1956.6 (r2=
0.9999) in, so as to obtain step 3) obtained by supernatant II in abiraterone concentration X, the X unit be μ g/ml；
Then through conversion, the abiraterone concentration in whole blood to be measured is obtained.
It is used as the improvement of the quantitative detecting method of abiraterone in the whole blood of the present invention：The step 1) in whole blood cracking
The preparation method of liquid is：Added in whole blood isometric cell pyrolysis liquid uniformly shake (in 100 ± 10r/min shake 5 ±
0.5 minute), obtain whole blood lysate；The formula of the cell pyrolysis liquid is：Ammonium chloride 8.29g, saleratus 1g and EDTA-2Na
37.2mg, is dissolved in 1000mL ultra-pure waters.
Reduction formula is X*20 μ L*10 ÷ 0.2mL=X；Therefore, the abiraterone concentration in whole blood to be measured is X.
In order to verify the accuracy and practicality of method of the invention, inventor has carried out following experiment：
1st, prepared by standard curve
Accurately weigh abiraterone standard items to be dissolved in chromatogram methanol, as storing solution, utilize mobile phase (that is, this hair
Mobile phase described in bright step 4) storing solution is diluted to 200.0,50.0,10.0,5.0,1.0,0.5 μ g/mL series marks respectively
Quasi- liquid, takes accurate addition 0.36mL blank whole blood lysates in 6 brace plug centrifuge tubes, then respectively add into above-mentioned each centrifuge tube
40 μ L above-mentioned standard liquid, produce the whole blood lysate of the standard items of abiraterone containing series concentration, i.e., after mixing：0.05,0.1,
0.5,1.0,5.0,20.0 μ g/mL series standard liquid.According to " sample-pretreating method and chromatostrip in foregoing invention content
Part " sample introduction is analyzed, with abiraterone medicine peak area (Y) for ordinate, is that abscissa makees standard curve with mass concentration (X),
Calibration curve equation and coefficient correlation (r) are obtained, i.e.,：Y=27524X+1956.6 (r2=0.9999).
2nd, the rate of recovery and precision
Prepare the high, medium and low μ g/mL of concentrations Plasma sample 0.05,0.5,5 in drug containing curve ranges and be used as quality-control sample
(QC), analyzed according to above-mentioned condition sample introduction, each concentration samples are repeated 5 times, with medicine peak area in sample and same concentrations
The ratio between standard sample peak area, calculates the rate of recovery under high, medium and low 3 kinds of concentration.
Prepare the high, medium and low μ g/mL of concentrations Plasma sample 0.05,0.5,5 in drug containing curve ranges and be used as quality-control sample
(QC), analyzed according to above-mentioned condition sample introduction, the peak area for comparing above sample in a few days 5 sample introductions and in the daytime 5 sample introductions changes
Amplitude, calculates the coefficient of variation (RSD) of in a few days and in the daytime peak area obtained by 5 sample introductions, so as to draw in a few days and day to day precision.
The rate of recovery and precision of abiraterone in the whole blood of table 2.
3rd, whole blood abiraterone assay after Oral Administration in Rats abiraterone
50 SPF grades of SD male rats are randomly divided into 10 groups, is slowly inserted in rat stomach, pressed using injecting type gastric perfusion needle
100mg/kg pours into abiraterone solution, without time person's of telling retention test.In 5 after administration, 10,20,30min, 1h, 2h, 4h, 8h,
12h and 24h tail veins take blood 0.2mL, are put into test tube of hepari centrifuge tube, add 0.2mL cell pyrolysis liquids, after concussion, make blood thin
Born of the same parents all crack.Accurate 0.4mL whole bloods lysate of drawing is analyzed according to sample introduction after above-mentioned pre-treating method and chromatographic condition processing,
The abiraterone peak area measured is substituted into above-mentioned standard curvilinear equation after sample introduction, Ah's bit in whole blood lysate sample is calculated
Imperial concentration, according to blood sampling time, draws blood concentration-time graph, sees Fig. 1.
The present invention has following technical advantage：
First, big change has been carried out to sample pre-treatments, using alkaline diatomite as filler, has been prepared into SPE small
Post, makes recovery of extraction be significantly improved；
Secondly, chromatographic column employs post and imitates highly efficient BDS C18 chromatographic columns, so that abiraterone separating degree is more
It is good so that sensitivity is significantly improved；
Finally, the present invention adds a certain proportion of acetonitrile in mobile phase, and it is about 2.5 to make mobile phase pH, so that
Eluting power is stronger, and biological impurities are eluted faster, more comprehensively in whole blood, thus it is noiseless to abiraterone chromatographic peak, to chromatogram
Post damage is smaller.
Brief description of the drawings
The embodiment to the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is abiraterone blood concentration --- time plot in whole blood of the invention；
Fig. 2 be comparative example 1 whole blood in abiraterone blood concentration --- time plot；
Fig. 3 be comparative example 2 whole blood in abiraterone blood concentration --- time plot；
Fig. 4 be comparative example 3-1 whole blood in abiraterone blood concentration --- time plot；
Fig. 5 be comparative example 3-2 whole blood in abiraterone blood concentration --- time plot；
Fig. 6 be comparative example 4 whole blood in abiraterone blood concentration --- time plot.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Abiraterone pre-treating method and quantitative detection means in embodiment 1, a kind of animal (people) whole blood, are carried out successively
Following steps：
1) the venous whole 0.2mL that, will be disengaged from animal body/human body is put into test tube of hepari centrifuge tube, is added 0.2mL cells and is split
Liquid is solved, (is shaken 5 minutes under 100r/min frequency) after concussion, makes haemocyte all cracking；Obtain whole blood lysate.
The formula of above-mentioned cell pyrolysis liquid is：Ammonium chloride 8.29g, saleratus 1g and EDTA-2Na37.2mg, are dissolved in
In 1000mL ultra-pure waters, stirring and dissolving.
The accurate whole blood lysate 0.4mL that draws is in centrifuge tube with cover, after vibration is mixed, accurate thereto to add acetic acid second
Ester 3mL, oscillator whirlpool (100r/min frequency) 2min, 8000r/min high speed centrifugation 5min, must be located at the second on upper strata respectively
Ethyl acetate layer, the residue positioned at lower floor.Take out whole upper strata ethyl acetate；Residue repeats above-mentioned shake with 2mL ethyl acetate again
Swing device whirlpool, high speed centrifugation (oscillator whirlpool 2min, 8000r/min high speed centrifugation 5min)；That is, residue repeats extraction one
It is secondary；
Merge the ethyl acetate (that is, merging ethyl acetate layer twice obtained by centrifugation) extracted twice in the centrifugation of sharp bottom glass
Guan Zhong, nitrogen stream is dried up in 45 DEG C of water-baths, and dried object 0.2mL 20% (volume %) acetonitrile solution redissolves, whirlpool
(100r/min frequency) is mixed after 1min, 18000r/min high speed centrifugations 5min, Aspirate supernatant I；The volume of this supernatant I
For 0.2mL；That is, the precipitation that high speed centrifugation is produced is very little, can be neglected；
2) alkaline diatomite (pH is 8.5 diatomite) 4g, is weighed to be put into glass chromatography column (10 × 200mm specifications),
Vibration handles 5min (in 100Hz oscillating column pumps)；
3) the 0.2mL supernatants I in and then by step 1) all adds steps 2) glass chromatography column in, it is first pure with 5mL
Water elution, is then eluted with 20% methanol (v/v) solution 5mL；Finally eluted with 5mL chromatograms methanol, collect chromatogram methanol institute right
The whole eluents answered, nitrogen stream is dried up in 45 DEG C of water-baths, and dried object 0.2mL 20% acetonitrile solution redissolves, whirlpool
(100r/min frequency) is mixed after 1min, 18000r/min high speed centrifugations 5min, and the μ L of Aspirate supernatant II 20 enter efficient liquid phase
Chromatographic system analysis；
Flow velocity during above-mentioned elution is controlled in 5mL/min.
4), Japanese Shimadzu shimadzu 20AT highly effective liquid phase chromatographic systems, Zhejiang University's intelligence reaches N2000 chromatographic work stations,
Chromatographic column uses Thermo Hypersil BDS C18 chromatographic columns (3 μm, 250mm × 4.6mm)；Mobile phase be acetonitrile-water-
0.01M sodium dihydrogen phosphates (pH 2.5)=60:5:35 (v/v), flow velocity 1.0mL/min, 40 DEG C of column temperature, ultraviolet detection wavelength
254nm, the μ L of sample size 20.
5), using step 4) obtained by abiraterone medicine peak area as Y, substitute into formula Y=27524X+1956.6 (r2=
0.9999) in, so as to obtain step 3) obtained by supernatant II in abiraterone concentration X, the X unit be μ g/ml；
The X is the abiraterone concentration in whole blood to be measured.
Comparative example 1, by the step 2 of embodiment 1) in alkaline diatomite change alumina packing into, do not make after loading chromatographic column
Vibration processing, remaining is equal to the step 1 of embodiment 1)~step 4).
It is in 0.05-20 μ g/mL concentration range internal standard directrix curves：Y=27465X -4627.8 (r2=0.9947).
The experiment of " rate of recovery and precision ", the rate of recovery and precision result such as table 3 below institute are carried out according to this comparative example 1-1
Show：
The rate of recovery and precision of abiraterone in the whole blood of table 3.
The experiment of " whole blood abiraterone assay after Oral Administration in Rats abiraterone ", gained are carried out according to this comparative example 1
Blood concentration-time graph as described in Figure 2.
Comparative example 2, by the step 3 of embodiment 1) in " 20% meoh eluate " change " 50% meoh eluate " into, remaining
It is equal to embodiment 1.
It is in 0.05-20 μ g/mL concentration range internal standard directrix curves：Y=24196X+5548.8 (r2=0.9985)
The experiment of " rate of recovery and precision ", the rate of recovery and precision result such as table 4 below institute are carried out according to this comparative example 2
Show：
The rate of recovery and precision of abiraterone in the whole blood of table 4.
The experiment of " whole blood abiraterone assay after Oral Administration in Rats abiraterone ", gained are carried out according to this comparative example 2
Blood concentration-time graph as described in Figure 3.
Comparative example 3-1, by the step 3 of embodiment 1) in chromatographic column by " BDS C18 chromatographic columns (and 3 μm, 250mm ×
4.6mm) " make into " ODS C18 chromatographic columns (5 μm, 250mm × 4.6mm) "；Remaining is equal to embodiment 1.
It is in 0.05-20 μ g/mL concentration range internal standard directrix curves：Y=23457X+4693.5 (r2=0.9973)
The experiment of " rate of recovery and precision ", the rate of recovery and precision result such as table 5 below institute are carried out according to this comparative example 3-1
Show：
The rate of recovery and precision of abiraterone in the whole blood of table 5.
The experiment of " whole blood abiraterone assay after Oral Administration in Rats abiraterone ", institute are carried out according to this comparative example 3-1
Blood concentration-the time graph obtained is as described in Figure 4.
Comparative example 3-2, by the step 3 of embodiment 1) in chromatographic column by " BDS C18 chromatographic columns (and 3 μm, 250mm ×
4.6mm) " make into " Betasil C18 chromatographic columns "；Remaining is equal to embodiment 1.
The experiment of " rate of recovery and precision ", the rate of recovery and precision result such as table 6 below institute are carried out according to this comparative example 3-2
Show：
The rate of recovery and precision of abiraterone in the whole blood of table 6.
The experiment of " whole blood abiraterone assay after Oral Administration in Rats abiraterone ", institute are carried out according to this comparative example 3-2
Blood concentration-the time graph obtained is as described in Figure 5.
Comparative example 4, by the step 3 of embodiment 1) in mobile phase by " acetonitrile-water -0.01M sodium dihydrogen phosphates (pH 2.5)=
60:5:35 (v/v) " make " acetonitrile-water -0.01M sodium dihydrogen phosphates (pH 3.5)=55 into:5:40(v/v)”；Remaining is equal to reality
Apply example 1.
It is in 0.05-20 μ g/mL concentration range internal standard directrix curves：Y=27537X -7944.7 (r2=0.9918).
The experiment of " rate of recovery and precision ", the rate of recovery and precision result such as table 7 below institute are carried out according to this comparative example 4
Show：
The rate of recovery and precision of abiraterone in the whole blood of table 7.
" whole blood abiraterone assay after Oral Administration in Rats abiraterone ", the blood of gained are carried out according to this comparative example 4
Concentration-time graph is as described in Figure 6.
Experiment 1, by following 3 parts of products to be tested：Whole blood A, whole blood B, whole blood C are detected according to the methods described of embodiment 1, are obtained
To abiraterone medicine peak area (Y), the abiraterone concentration substituted into formula Y=27524X+1956.6 acquisition supernatants
(X), then following reduction formula is substituted into：X*20 μ L*10 ÷ 0.2mL, obtain the abiraterone concentration in whole blood to be measured；
Acquired results such as table 8 below；
Table 8
Confirmatory experiment, according to HPLC-MS methods whole blood is detected, abiraterone concentration such as following table in the whole blood of gained
9：
Table 9
| Abiraterone concentration in whole blood | |
| Whole blood A | 0.022 |
| Whole blood B | 0.33 |
| Whole blood C | 2.78 |
Finally, in addition it is also necessary to it is noted that listed above is only the specific embodiment of abiraterone in the present invention.Obviously,
The invention is not restricted to above example, there can also be many deformations.One of ordinary skill in the art can be from disclosed by the invention
All deformations that content is directly exported or associated, are considered as protection scope of the present invention.
Claims (5)
1. the quantitative detecting method of abiraterone in whole blood, it is characterized in that：Using whole blood as object to be measured, following walk is carried out successively
Suddenly：
1) whole blood lysate 0.4mL, is drawn in lidded container, adds ethyl acetate 3mL, oscillator 2 ± 0.2min of whirlpool,
8000 ± 500r/min, 5 ± 0.5min of high speed centrifugation, must be located at ethyl acetate layer, the residue positioned at lower floor on upper strata respectively；
2mL ethyl acetate is added in residue and repeats above-mentioned oscillator whirlpool, high speed centrifugation；
Merge the ethyl acetate layer twice obtained by centrifugation；Nitrogen stream is dried up in 45 ± 1 DEG C of water-baths, and the dried object of gained is used
0.2mL 20% acetonitrile solution redissolves, and whirlpool mixes 1 ± 0.2min, 18000 ± 500r/min high speed centrifugations, 5 ± 0.5min
Afterwards, Aspirate supernatant I；
2), weigh alkaline diatomite 4g to be put into glass chromatography column, 5 ± 0.5min of vibration processing；
3) supernatant I in and then by step 1) all adds steps 2) glass chromatography column in, first eluted with 5mL pure water, so
Eluted afterwards with 20% methanol aqueous solution 5mL；Finally eluted with 5mL chromatograms methanol, collect whole elutions corresponding to chromatogram methanol
Liquid, nitrogen stream is dried up in 45 ± 1 DEG C of water-baths, and dried object 0.2mL 20% acetonitrile solution redissolves, whirlpool mixing 1 ±
After 5 ± 0.5min of 0.2min, 18000 ± 500r/min high speed centrifugation, Aspirate supernatant II carries out follow-up high performance liquid chromatography
Network analysis；
4), chromatographic column uses Thermo Hypersil BDS C18 chromatographic columns (3 μm, 250mm × 4.6mm)；Mobile phase is second
Nitrile-water -0.01M sodium dihydrogen phosphate=60:5:35 (v/v), flow velocity 1.0mL/min, 40 DEG C of column temperature, ultraviolet detection wavelength 254nm,
The μ L of sample size 20；
5), using step 4) obtained by abiraterone medicine peak area as Y, substitute into formula Y=27524X+1956.6, so as to obtain
Step 3) obtained by supernatant II in abiraterone concentration X, the X unit be μ g/ml；
Then through conversion, the abiraterone concentration in whole blood to be measured is obtained.
2. the quantitative detecting method of abiraterone in whole blood according to claim 1, it is characterized in that：The step 1) in
The preparation method of whole blood lysate is：Isometric cell pyrolysis liquid is added in whole blood uniformly to shake, and obtains whole blood lysate.
3. the quantitative detecting method of abiraterone in whole blood according to claim 2, it is characterized in that：The step 1) in：
Shaken 5 ± 0.5 minutes in 100 ± 10r/min.
4. according to the quantitative detecting method of abiraterone in any described whole blood of claims 1 to 3, it is characterized in that：It is to be measured complete
Abiraterone concentration in blood is X.
5. the quantitative detecting method of abiraterone in whole blood according to claim 4, it is characterized in that：
The formula of the cell pyrolysis liquid is：Ammonium chloride 8.29g, saleratus 1g and EDTA-2Na 37.2mg, are dissolved in 1000mL
In ultra-pure water.
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