CN106841420A - A kind of method that LC MS/MS determine Ly 7u concentration in rat plasma - Google Patents
A kind of method that LC MS/MS determine Ly 7u concentration in rat plasma Download PDFInfo
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- CN106841420A CN106841420A CN201611255758.5A CN201611255758A CN106841420A CN 106841420 A CN106841420 A CN 106841420A CN 201611255758 A CN201611255758 A CN 201611255758A CN 106841420 A CN106841420 A CN 106841420A
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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
Abstract
The invention discloses a kind of method that LC MS/MS determine Ly 7u concentration in rat plasma, comprise the following steps：A series of Ly 7u and internal standard Ly 7z standard working solutions are prepared first, liquid-liquid extraction extracting method pre-treatment rat blank plasma is taken, is analyzed using LC MS/MS, set up standard curve；Then sample to be tested blood plasma is weighed, internal standard is added, using liquid-liquid extraction extracting method pre-treatment, is analyzed using LC MS/MS, obtain the chromatogram of each sample, the concentration of sample blood plasma is calculated using standard curve.Plasma sample pre-treating method of the invention is easy to operate, efficient stable, process time is short, use extractant safety non-toxic, detection technique sensitivity is high, and analysis time is short, and the degree of accuracy and precision are high, can be that the oral administration biaavailability of the compound and new drug preclinical study provide technical support the need for Ly 7u pharmcokinetic evaluations can well be met.
Description
Technical field
The invention belongs to pharmacokinetic analysis technical field.More particularly, to a kind of LC-MS/MS (liquid chromatogram matter
Spectrum GC-MS) determine rat plasma in Ly-7u concentration method.
Background technology
Aurora kinase (aurora kinases) is the important protein kinase of a class during cell mitogen, in cell
Mitosis in play important regulating and controlling effect, in recent years, correlative study find aurora kinase in tumour is formed have weight
Function is wanted, therefore a series of kinase inhibitors are developed research.
Compound L y-7u is the achievement in research of the present inventor team early stage, and its structural formula is as follows.In vitro study shows
Show, the compound has good antitumor activity, and with Aurora A/B kinase inhibiting activities and selectivity, with fine
Application prospect, the current compound has been in preclinical study conceptual phase.
Pharmcokinetic evaluation is that quantitative study medicine absorbs, is distributed, being metabolized and total quantity control in vivo, and is used
Mathematical principle and method illustrate the rule that blood concentration is changed over time.With pharmaceutical chemical development and level of human health
Improve constantly, to the requirement more and more higher of the pharmacokinetic property of medicine：Judge an application prospect for medicine particularly city
Field prospect, is not merely strong curative effect, and toxic and side effect is small；More to possess good pharmacokinetic property.And medicine is in blood plasma
The detection technique of concentration is the important prerequisite technology of pharmacokinetic analysis assessment.Therefore, commented to meet Ly-7u pharmacokinetics
The need for estimating, a kind of sensitivity Ly-7u PC detection methods high, reliable and stable are to study its oral administration biaavailability
Basis.
The content of the invention
Primary and foremost purpose of the invention is to provide a kind of method for detecting Ly-7u concentration in rat plasma, is a kind of quick, spirit
Sensitivity is high, the degree of accuracy and precision liquid chromatography mass high are combined detection method, the need for meeting Ly-7u preclinical studies,
Technical support can be provided for the new drug preclinical study of the compound.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of method that LC-MS/MS determines Ly-7u concentration in rat plasma, comprises the following steps：
S1. standard working solution is prepared：Precision weighs Ly-7u and internal standard Ly-7z powder, is dissolved with methanol aqueous solution respectively,
And it is diluted to standard working solution；
S2. standard curve is set up：Precision measures rat blank plasma, adds a series of Ly-7u standard working solutions, adds interior
Mark Ly-7z standard working solutions, using liquid-liquid extraction extracting method pre-treatment, are analyzed using LC-MS/MS afterwards, obtain various kinds
This chromatogram, with determinand and internal standard peak area ratio as abscissa, standard curve is set up by ordinate of testing concentration；
S3. sample to be tested plasma treatment：Precision measures rat plasma after administration Ly-7u, internal standard is added, using liquid-liquid extraction
Extracting method pre-treatment；
S4. it is analyzed using LC-MS/MS, obtains the chromatogram of each sample, sample blood plasma is calculated using standard curve
Concentration.
Wherein it is preferred to, the volume ratio composition of methanol aqueous solution described in step S1 is methyl alcohol：Water=90：10.
Preferably, also need to be dissolved by 5~15min of ultrasound after being dissolved with methanol aqueous solution described in step S1.
It is highly preferred that being that ultrasonic 10min is dissolved.
Preferably, after Ly-7u standard working solutions are added in step S2 and internal standard Ly-7z standard working solutions are added, it is required for
Light whirlpool mixes.
Preferably, ethyl acetate is selected in liquid-liquid extraction described in step S2.
It is highly preferred that liquid-liquid extraction extracting method pre-treatment is described in step S2 or S3：1mL ethyl acetate is added, is vortexed
2min, stand 10min, 15000rpm centrifugation 10min, Aspirate supernatant, at room temperature vacuum volatilize, add redissolve liquid dissolving, whirlpool
Rotation 1.5min, 15000rpm centrifugation 5min；The volume ratio for redissolving liquid constitutes and is：Methyl alcohol：0.1% formic acid water=9：1, wherein
Also contain 5mM ammonium acetates.
Preferably, the chromatographic condition setting of LC-MS/MS described in step S2 or S4 is as follows：Mobile phase (contains for methyl alcohol (A)/water
The ammonium acetate of 0.1% formic acid and 5mM) (B)；Gradient elution (0-1min, 5%-90%A；1-4min, 90%-90%A；4–
4.1min, 90% -5%A；4.1-5min, 90%-90%A)；
Flow velocity 0.3mLmin-1；The μ L of sample size 2；25 DEG C of column temperature；MRM patterns：Ly-7u：m/z 410→m/z353；Ly-
7z：m/z 457→m/z 368.
Preferably, the quantitative daughter ion of Ly-7u is 353m/z in LC-MS/MS described in step S2 or S4, interior scalar quantity from
Son is 368m/z.
Preferably, all water used are ultra-pure water, and all organic reagents are HPLC grades.
Preferably, the LC-MS/MS is UHPLC-MS/MS.
Furthermore it is preferred that rat blank plasma in step S2：Ly-7u standard working solutions：Internal standard Ly-7z standard working solutions
Volume ratio=9：1：4.
It is highly preferred that being to take 45 μ l rat blank plasmas, 5 μ L Ly-7u standard working solutions and 20 μ L inner mark solutions are added,
Ethyl acetate consumption is 1mL when the liquid-liquid extraction is extracted.
Particularly preferably, liquid-liquid extraction method pre-treatment is described in step S2：
1mL ethyl acetate, vortex 2min is added to stand 10min, 15000rpm centrifugation 10min, draw 800 μ L of supernatant liquid
Into another clean 1.5mL centrifuge tubes, vacuum is volatilized at room temperature, and [methyl alcohol/0.1% formic acid water (contains 5mM to add 200 μ L to redissolve liquid
Ammonium acetate)=9:1, v/v] dissolve, vortex 1.5min, 15000rpm centrifugation 5min.
The invention has the advantages that：
The present invention provides a kind of method that LC-MS/MS determines Ly-7u concentration in rat plasma first, to plasma sample
Pre-treating method is probed into and optimized so that the method for being used is easy to operate, and efficient stable, process time is short, uses extraction
Take solvent safety nontoxic.
Meanwhile, the present invention to chromatographic condition due to rationally being set so that using to be measured obtained by the method for the present invention
Thing and internal standard chromatogram are reliable and stable, and detection technique sensitivity is high, and analysis time is short, and the degree of accuracy and precision are high, can be fine
Meet Ly-7u pharmcokinetic evaluations the need for.
The LC-MS/MS detection methods of Ly-7u in the rat plasma that the present invention sets up, can be applied to research Ly-7u rats mouthful
Bioavilability is taken, the purpose that the method meets Ly-7u preclinical studies is reached.
Brief description of the drawings
Fig. 1 is the Ly-7u ion massspectrum figures that the embodiment of the present invention 1 is set up；
Fig. 2 is the standard curve for determining Ly-7u concentration in rat plasma that the embodiment of the present invention 1 is set up；
Fig. 3 is the sample chromatogram of the LC-MS/MS methods that the embodiment of the present invention 1 is set up.
Fig. 4 is the sample chromatogram of the LC-MS/MS methods that the embodiment of the present invention 2 is set up.
Fig. 5 is Drug-time curve figure after the measure rat single oral that the embodiment of the present invention 3 is set up and intravenous Ly-7u preparations；
Wherein, ordinate is the logarithm value of Ly-7u concentration (C).
Specific embodiment
The present invention, but embodiment are further illustrated below in conjunction with Figure of description and specific embodiment not to the present invention
Limit in any form.Unless stated otherwise, reagent, the method and apparatus that the present invention is used are for the art is routinely tried
Agent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are purchased in market.
Embodiment 1
1st, the standard curve for determining Ly-7u concentration in rat plasma is set up：
(1) standard working solution is prepared：
Precision weighs Ly-7u standard items 3.00mg in 10ml volumetric flasks, methanol/water (9:1) constant volume, ultrasonic 10min is molten
Solution, obtains final product the standard reserving solution of 300 μ g/mL, is placed in -20 DEG C of preservations.Prepare concentration be respectively 50,000,40,000,20,000,
10,000th, 5000,2000,500,100,20,5, the Ly-7u standard working solutions of 2.5ng/mL.
Precision weighs internal standard Ly-7z standard items 5.00mg in 5ml volumetric flasks, methanol/water (9:1) constant volume, ultrasonic 10min
Dissolving, obtains final product the standard reserving solution of 1mg/mL, is placed in -20 DEG C of preservations.It is diluted to (methanol/water=50/50, v/v) using preceding
500ng/ml standard working solutions.
The structural formula of internal standard Ly-7z is as follows：
(2) standard curve is set up：
1) the μ l of standard working solution 5 are taken and adds 45 μ l blank rat plasmas, light whirlpool mixes, and adds the μ L of inner mark solution 20, light whirlpool
Mix, add 1mL ethyl acetate, vortex 2min to stand 10min, 15000rpm centrifugation 10min, draw 800 μ L of supernatant liquid to another
In one clean 1.5mL centrifuge tubes, vacuum is volatilized at room temperature, adds 200 μ L to redissolve liquid [methyl alcohol/0.1% formic acid water (acetic acid containing 5mM
Ammonium)=9:1, v/v] dissolve, vortex 1.5min, 15000rpm centrifugation 5min.The μ l of supernatant 120 move into sample injection bottle, and the μ L of sample introduction 2 enter
Row UHPLC-MS/MS is analyzed.
2) it is accurate to draw the μ L sample introductions of need testing solution 120, eluted using following chromatographic condition：
Chromatographic column hypersil gold C18 chromatographic columns (2.1mm × 100mm, 1.9 μm；Thremo,USA)；
Mobile phase is methyl alcohol (A)/water (ammonium acetate containing 0.1% formic acid and 5mM) (B), gradient elution (0-1min, 5%-
90%A；1-4min, 90%-90%A；4-4.1min, 90%-5%A；4.1-5min, 90%-90%A)；
Flow velocity 0.3mLmin-1；The μ L of sample size 2；25 DEG C of column temperature；MRM patterns：Ly-7u：m/z 410→m/z353；Ly-
7z：m/z 457→m/z 368.Result is as shown in Figure 1.
3) LC-MS/MS analyses are carried out, with the ratio between the peak area of the peak area of the Ly-7u of various concentrations and internal standard Ly-7z Y
It is ordinate, linear regression is carried out by abscissa of the blood concentration C of Ly-7u, obtains regression equation and R2.With signal to noise ratio S/N=3
Meter, obtains the minimal detectable concentration of the method.Standard curve result is as shown in Figure 2.
2nd, the sample chromatogram of the LC-MS/MS methods set up
The μ L of rat plasma sample 50, are placed in 1.5mL EP pipes, add the corresponding μ L of inner mark solution 20, add 1mL acetic acid second
Ester, vortex 2min stands 10min, 15000rpm centrifugation 10min, draws 800 μ L of supernatant liquid to another clean 1.5mL centrifuge tubes
In, vacuum is volatilized at room temperature, adds 200 μ L to redissolve liquid [methyl alcohol/0.1%/formic acid water (ammonium acetate containing 5mM)=9:1, v/v] it is molten
Solution, vortex 1.5min, 15000rpm centrifugation 5min, the μ l of supernatant 120 move into sample injection bottle, and the μ L of sample introduction 2 carry out UHPLC-MS/MS points
Analysis.
The blood concentration C that standard curve calculates Ly-7u is substituted into the ratio between the peak area of Ly-7u and interior target peak area Y.
2nd, sample chromatogram result is as shown in Figure 3.Result shows that determinand and internal standard peak shape are smooth symmetrical in chromatogram,
Noiseless peak, chromatogram is reliable and stable, and appearance time is respectively 2.94min and 2.99min, and analysis time is short.Show this measure
Method is efficient, stabilization, the need for can well meeting Ly-7u pharmcokinetic evaluations.
The mark-on pattern detection of embodiment 2
1st, spiked plasma Sample pretreatment
(1) from about 300 μ L of blank rat orbital venous plexus blood sampling.Blood sample is placed in test tube of hepari EP pipes, whole blood warp
4000rpm centrifugations 5min draws upper plasma, i.e. blank rat plasma, by 100 μ L packing after -80 DEG C of storages.
Blank rat plasma will be added outside Ly-7u, obtain Ly-7u concentration be 0.5,50,500, the mark-on blood of 4000ng/mL
Slurry samples.
(2) the μ L of spiked plasma sample 100 are taken, after being processed according to the Sample pretreatment of embodiment 1, the μ L of supernatant 80 shiftings is taken
In entering sample injection bottle.
2nd, spiked plasma pattern detection
The μ L of sample introduction 10 carry out UPLC-MS/MS analyses, and condition is with embodiment 1.Plasma sample concentration is calculated according to standard curve
0.4 ± 0.05,49.1 ± 1.4,505.0 ± 11.2,3923.3 ± 158.7ng/mL, wherein Ly-7u concentration for 50ng/mL sample
This chromatogram is as shown in Figure 4.
Show that this assay method can accurately determine the concentration of Ly-7u in analysis blood plasma, and determinand in chromatogram very much
Smooth with internal standard peak shape symmetrical, noiseless peak, chromatogram is reliable and stable, and method is efficient, stabilization, can well meet Ly-7u medicines
The need for dynamic assessment.
The pattern detection of embodiment 3
1st, the absolute bioavailability research plasma sample pre-treatment of the oral Ly-7u preparations of SD rats single oral gavage
(1) after SD rats single oral gavage, intravenous injection administration, according to experimental design sampling time scheme, from orbital vein
About 300 μ L of clump blood sampling.Blood sample is placed in test tube of hepari EP pipes, and whole blood draws upper plasma through 4000rpm centrifugations 5min, by 100 μ
L packing is after -80 DEG C of storages to measure.
(2) the μ L of rat plasma sample 100, pre-treatment is carried out with the Sample pretreatment method described in embodiment 1, takes supernatant 80
μ L are moved into sample injection bottle.
2nd, the absolute bioavailability research plasma sample detection of the oral Ly-7u preparations of SD rats single oral gavage
The μ L of sample introduction 10 carry out UPLC-MS/MS analyses, and it is dense that condition calculates plasma sample with embodiment 1, according to standard curve
Degree.
Result shows that the concentration of each time point Ly-7u is as shown in Figure 5 in oral, intravenous group SD rat plasmas.
Claims (10)
1. a kind of method that LC-MS/MS determines Ly-7u concentration in rat plasma, it is characterised in that comprise the following steps：
S1. standard working solution is prepared：Precision weighs Ly-7u and internal standard Ly-7z powder, is dissolved with methanol aqueous solution respectively, and dilute
It is interpreted into standard working solution；
S2. standard curve is set up：Precision measures rat blank plasma, adds a series of Ly-7u standard working solutions, adds internal standard
Ly-7z standard working solutions, using liquid-liquid extraction extracting method pre-treatment, are analyzed using LC-MS/MS afterwards, obtain each sample
Chromatogram, with determinand and internal standard peak area ratio as abscissa, standard curve is set up by ordinate of testing concentration；
S3. sample to be tested plasma treatment：Precision measures rat plasma after administration Ly-7u, adds internal standard, is carried using liquid-liquid extraction
Take method pre-treatment；
S4. it is analyzed using LC-MS/MS, obtains the chromatogram of each sample, sample blood plasma is calculated using standard curve
Concentration.
2. method according to claim 1, it is characterised in that the volume ratio composition of methanol aqueous solution described in step S1 is first
Alcohol：Water=90：10.
3. method according to claim 1, it is characterised in that also needed after being dissolved with methanol aqueous solution described in step S1 by
5~15min of ultrasound is dissolved.
4. method according to claim 1, it is characterised in that in Ly-7u standard working solutions being added in step S2 and being added
After mark Ly-7z standard working solutions, it is required for light whirlpool to mix.
5. method according to claim 1, it is characterised in that ethyl acetate is selected in liquid-liquid extraction described in step S2.
6. method according to claim 1, it is characterised in that locate before liquid-liquid extraction extracting method described in step S2 or S3
Manage and be：Ethyl acetate is added, be vortexed 2 min, standing 10 min, 15000 rpm 10 min of centrifugation, Aspirate supernatant, at room temperature
Vacuum is volatilized, and is added and is redissolved liquid dissolving, and be vortexed 1.5 min, and 15000 rpm are centrifuged 5 min；The volume ratio composition for redissolving liquid
For：Methyl alcohol：0.1% formic acid water=9：1, wherein also containing 5mM ammonium acetates.
7. method according to claim 1, it is characterised in that the chromatographic condition of LC-MS/MS sets described in step S2 or S4
It is fixed as follows：Mobile phase is methyl alcohol (A)/water（Ammonium acetate containing 0.1% formic acid and 5 mM）(B)；Gradient elution (0-1 min, 5%-
90% A; 1–4 min, 90%–90% A; 4–4.1 min, 90%–5% A; 4.1–5 min, 90%–90% A)；
The mLmin of flow velocity 0.3-1；The μ L of sample size 2；25 DEG C of column temperature；MRM patterns：Ly-7u：m/z 410→m/z 353；Ly-
7z：m/z 457→m/z 368.
8. method according to claim 1, it is characterised in that Ly-7u's quantifies in LC-MS/MS described in step S2 or S4
Daughter ion is 353m/z, and interior scalar quantity daughter ion is 368 m/z.
9. method according to claim 1, it is characterised in that all water used are ultra-pure water, all organic reagents
It is HPLC grades.
10. method according to claim 1, it is characterised in that the LC-MS/MS is UHPLC-MS/MS.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| WO2012158658A1 (en) * | 2011-05-16 | 2012-11-22 | OSI Pharmaceuticals, LLC | Fused bicyclic kinase inhibitors |
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| CN101360740A (en) * | 2005-11-16 | 2009-02-04 | 沃泰克斯药物股份有限公司 | Aminopyrimidines useful as kinase inhibitors |
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Application publication date: 20170613 |