CN104133004A - Determination method of concentration of 2,3-isatin in plasma sample - Google Patents
Determination method of concentration of 2,3-isatin in plasma sample Download PDFInfo
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Abstract
The invention relates to a determination method of concentration of 2,3-isatin in a plasma sample. The determination method includes following steps: (1) pre-treatment of the plasma sample: mixing an animal blood sample, which is collected after the 2,3-isatin being given to the animal, with an internal standard substance methanol solution at 0-4 DEG C according to a volume ratio of 1:5, carrying out a vortex process for 30-90 seconds and carrying out a centrifugal process to obtain a supernate; and (2) a detection step: detecting the pre-treated plasma sample through combination of liquid chromatography and mass spectrum. Liquid chromatography conditions includes: a chromatographic column is Ascentis, RP-Amide, 5[mu]m, 50*2.1mm; a chromatography mobile phase A is a water solution containing 5 mM of ammonium formate and 5 mM of formic acid and a chromatography mobile phase b is methonal, wherein the volume percentages of the mobile phase A and the mobile phase B are respectively 55% and 45%. Mass spectrum conditions includes: Type of mass spectrum scanning is positive-ion multi-reaction monitoring; an ion source is an electric-spraying ion source; a pressure of atomized gas is 50 psi; a gas velocity is 9L/min; a capillary voltage is 4500 V; a ion source temperature is 350 DEG C; and a quantifying operation is carried out through an internal standard method.
Description
Technical field
The invention belongs to biological medicine detection field, relate to the assay method of the concentration in plasma sample in a kind of biosome of endogenous reactive compound 2,3-indolinedione.
Background technology
2,3-indolinedione (2,3-Indolinedione, Isatin, ISA), has another name called isatin, is a kind of endogenous active factors of in recent years finding to be present in humans and animals body, has multiple biologically active.
Liquid chromatography and mass spectrometric hyphenated technique (LC-MS), liquid chromatography-atmospheric pressure ionization mass spectrometry coupling technique (LC-API-MS) especially wherein, is the first-selection of trace materials in present analysis biological sample.It has had the advantage of liquid chromatography and two kinds of technology of mass spectrum concurrently, has high sensitivity and selectivity,, and greatly shorten analysis time.
At present, have been reported 2, 3-istain assay method has ECL, GC-MS, the method such as LC-MS and HPLC, the most sample pre-treatments of these methods is complicated, some need to carry out multistep purification process to biological sample, experimental implementation process is more loaded down with trivial details, and sample analysis time is longer, be used to 2, in the interior pharmacokinetic of body of 3-istain, the analysis test method of large quantities of biological samples also seldom, and there is above problem, therefore a large amount of samples that produce in processing pharmacokinetic just need to be developed high-throughout rapid analysis, particularly sample pre-treatments is easy, the rapid analysis that detection means is sensitive.For example Chinese patent CN102735764A discloses the assay method of Content of Ribavirin in a kind of blood plasma, and the method is used comparatively easy blood plasma preprocess method, and uses LC-MS method to detect, and sensitivity is higher.But the fast and convenient sensitive assay method about 2,3-indolinedione concentration in plasma sample also rarely has report at present.
Summary of the invention
Problem in the biological sample concentration analysis existing in Preclinical metabolism and pharmacokinetics study based on 2,3-indolinedione, the object of the present invention is to provide a kind of LC-MS analytical approach of 2,3-indolinedione concentration in a kind of high flux Rapid Determination of Plasma.
At this, the invention provides the assay method of the concentration of 2,3-indolinedione in a kind of plasma sample, comprising:
Plasma sample pre-treatment step:
At 0～4 ℃, for mixing animal, 1:5 gives the plasma sample (containing the plasma sample of 2,3-indolinedione) and the inner mark solution that after 2,3-indolinedione, gather by volume, vortex is centrifuging and taking supernatant after 30～90 seconds, the flat methanol solution of Kui sulphur that wherein said inner mark solution is 10ng/mL; Detecting step plasma sample through pre-treatment being detected by liquid chromatograph mass spectrography, wherein
Liquid phase chromatogram condition comprises: chromatographic column is Ascentis, RP-Amide, 5 μ m, 50 * 2.1mm; Chromatogram flow phase A is 5mM ammonium formate/5mM aqueous formic acid, and Mobile phase B is methyl alcohol, and the percent by volume of mobile phase A and B is respectively 55% and 45%;
Mass spectrum condition comprises: scanning of the mass spectrum type is positive ion multiple-reaction monitoring; Ion gun is electric spray ion source; Atomization gas pressure is 50psi; Gas flow rate is 9L/min; Capillary voltage is 4500V; Ion source temperature is 350 ℃, inner mark method ration.
The present invention processes plasma sample by the Methanol Protein precipitation method, by the concentration of 2,3-indolinedione in liquid chromatography mass coupling instrument analysed for plasma sample.Therefore, method of the present invention have advantages of that pre-treatment is easy, detection means sensitive single-minded, detection speed is fast.The accuracy of method of the present invention, precision and detectability meet biological sample analysis methodology checking requirement, can meet the concentration determination needs of plasma sample in the pharmacokinetic of 2,3-indolinedione.
In described plasma sample pre-treatment step, described internal standard compound methanol solution is preferably the flat methanol solution of Kui sulphur of 10ng/mL.Again, the described centrifugal centrifugal 5min of speed being preferably with 15000r/min.
Again, in described detecting step, described liquid phase chromatogram condition also comprises: column temperature is 40 ℃; Auto injection actuator temperature is 6 ℃; Washing pin liquid is isopropanol water solution, and wherein, the volume ratio of isopropyl alcohol and water is 1:1; Be 2 minutes analysis time, and flow velocity is 400 μ L/min, and sample size is 5 μ L.
In described detecting step, described mass spectrum condition also comprises: the ion pair for quantitative test is: 2,3-indolinedione: 148 → 65.1; Kui sulphur is flat: 384.1 → 253.1.
In the present invention, described plasma sample is mammiferous blood plasma.
In one example, described plasma sample is that animal gives the fresh plasma gathering after 2,3-indolinedione sample.
In another example, described plasma sample is that animal gives 2, the plasma sample gathering after 3-istain is deposited in the deposit plasma sample of-80 ℃ of refrigerators, described plasma sample pre-treatment step first takes out described deposit plasma sample before being also included in and mixing with described internal standard compound methanol solution from-80 ℃ of refrigerators, after naturally melting at 0～4 ℃, whirlpool is 20～40 seconds.
In the present invention, the lowest detection of described assay method is limited to 10ng/mL.Can be satisfied with the detection of low content 2,3-indolinedione.
In the present invention, the relative error of described assay method and relative standard deviation are all in 20%, and reappearance and reliability are higher, have higher stability.
Accompanying drawing explanation
Fig. 1 is selectivity sample chromatogram figure in rat plasma, wherein Fig. 1 (a) is 2 of blank plasma, 3-istain chromatogram, Fig. 1 (b) is the flat chromatogram of Kui sulphur of blank plasma, Fig. 1 (c) is that blank plasma adds interior target 2,3-istain chromatogram, Fig. 1 (d) is that blank plasma adds the flat chromatogram of interior target Kui sulphur;
Fig. 2 is minimum lower limit of quantitation sample chromatogram figure in rat plasma, and wherein Fig. 2 (a) is 2,3-indolinedione chromatogram, and Fig. 2 (b) is the flat chromatogram of Kui sulphur;
Fig. 3 is rat plasma system residual sample chromatogram, and wherein Fig. 3 (a) is 2,3-indolinedione chromatogram, and Fig. 3 (b) is the flat chromatogram of Kui sulphur;
Fig. 4 is selectivity sample chromatogram figure in Beagle dog (beasle dog) blood plasma, wherein Fig. 4 (a) is 2 of blank plasma, 3-istain chromatogram, Fig. 4 (b) blank plasma be the flat chromatogram of Kui sulphur, Fig. 4 (c) is that blank plasma adds interior target 2,3-istain chromatogram, Fig. 4 (d) is that blank plasma adds the flat chromatogram of interior target Kui sulphur;
Fig. 5 is minimum lower limit of quantitation sample chromatogram figure in Beagle dog (beasle dog) blood plasma, and wherein Fig. 5 (a) is 2,3-indolinedione chromatogram, and Fig. 5 (b) is the flat chromatogram of Kui sulphur;
Fig. 6 is Beagle dog (beasle dog) blood plasma system residual sample chromatogram, and wherein Fig. 6 (a) is 2,3-indolinedione chromatogram, and Fig. 6 (b) is the flat chromatogram of Kui sulphur.
Embodiment
Below, with reference to accompanying drawing, and further illustrate with the following embodiments the present invention.Should understand these embodiments and embodiment only for the present invention is described, and be not used in restriction the present invention.
A kind of LC-MS analytical approach that the invention provides 2,3-indolinedione concentration in a kind of high flux Rapid Determination of Plasma, relates to: the albumen precipitation pre-treating method of (1) plasma sample; (2) preparation of 2,3-indolinedione typical curve in plasma sample; (3) method for separating liquid phase chromatography; (4) Mass Spectrometer Method method; (5) calculating of result.
The present invention processes plasma sample by the Methanol Protein precipitation method, by the concentration of 2,3-indolinedione in liquid chromatography mass coupling instrument analysed for plasma sample.The accuracy of this method, precision and detectability meet biological sample analysis methodology checking requirement, can meet the concentration determination needs of plasma sample in the pharmacokinetic of 2,3-indolinedione.
As an example, method of the present invention can relate to following aspect.
(1) the albumen precipitation pre-treatment of plasma sample: get 50 μ L plasma samples to 1.5mL centrifuge tube, (for example add 250 μ L inner mark solutions, the flat methanol solution of 10ng/mL Kui sulphur), vortex centrifugal after 30～90 seconds (for example 15000r/min, centrifugal 5 minutes 4 ℃); Get in supernatant 100 μ L to Agilent chromatogram sample introduction bottles.Whole pretreatment process carries out under 4 ℃ (or (being about 0～4 ℃) on ice).Plasma sample can be the fresh plasma for example, taking out from mammal (rat or dog), it is directly placed in and carries out above-mentioned pre-treatment on ice, also the fresh plasma taking out from mammal can be kept in advance in-80 ℃ of refrigerators, when needing use, from-80 ℃ of refrigerators, take out sample, 4 ℃ (or (being about 0～4 ℃) on ice), naturally dissolve rear vortex and mix with inner mark solution again after 30 seconds.
(2) preparation of typical curve sample:
A) 2, the preparation of 3-istain storing solution (Stock A): precision takes 2,3-istain standard items 1.30mg, be placed in 5mL volumetric flask, with methyl alcohol, dissolve and be diluted to scale, shaking up, being mixed with mass concentration is 2 of 260 μ g/mL, 3-istain storing solution (Stock A), is stored in 4 ℃ of refrigerators stand-by;
B) preparation of 2,3-indolinedione standard solution: 2,3-indolinedione storing solution (Stock A) is diluted to 200 μ g/mL with methyl alcohol, then use methyl alcohol stepwise dilution, being mixed with 2,3-indolinedione concentration is 100,40,20,10,4, the standard solution of 2,0.4 μ g/mL, as table 1, wherein the CSS in table 1 represents that typical curve sample adds solution (Calibration Standard Spiking Solution)
Table 1, the preparation of 2,3-indolinedione standard solution:
C) preparation of 2,3-indolinedione typical curve sample:
By the 2,3-indolinedione standard solution of step b) gained each 5 μ L are parallel joins in 195 μ L blank plasma matrix, make 2,3-istain concentration is 5000,2500,1000,500,250,100,50, the typical curve sample of 10ng/mL, as table 2, wherein STD represents typical curve sample (Calibration Standard Sample)
Table 2,2,3-indolinedione typical curve sample preparation:
(3) preparation of quality-control sample:
A) 2, the preparation of 3-istain storing solution (Stock B): precision takes 2,3-istain standard items 1.355mg, is placed in 5mL volumetric flask, with methyl alcohol, dissolves and is diluted to scale, shake up, being mixed with mass concentration is the 2,3-indolinedione storing solution (Stock B) of 271 μ g/mL, as 2,3-istain Quality Control storing solution, is stored in 4 ℃ of refrigerators stand-by;
B) preparation of 2,3-indolinedione Quality Control solution: by 320 μ L2,3-istain storing solution (Stock B) and 222 μ L methyl alcohol mix, and obtaining 2,3-indolinedione concentration is the high quality-control sample annex solution (QSS-High) of 160 μ g/mL.By methyl alcohol dilution for high quality-control sample annex solution (QSS-High), being mixed with 2,3-indolinedione concentration is the Quality Control solution of 16 and 3.2 μ g/mL, and as table 3, wherein QSS represents that quality-control sample adds solution (Quality Control Spiking Solution),
Table 3, Quality Control solution preparation:
C) 2, the preparation of 3-istain quality-control sample: by the quality control standard solution of step b) gained each 50 μ L are parallel joins in 1950 μ L blank plasma matrix, make 2,3-istain concentration is 4000,400 and the quality-control sample of 80ng/mL, as table 4, wherein QC represents quality-control sample (Quality Control Sample).Quality-control sample is sub-packed in 1.5mL centrifuge tube by every part of 200 μ L, is stored in-80 ℃ of refrigerators,
Table 4: quality-control sample preparation:
(4) the flat storing solution of Kui sulphur (IS Stock A, interior mark) preparation: precision takes the flat standard items 2.72mg of Kui sulphur, be placed in 5mL volumetric flask, with methyl alcohol, dissolve and be diluted to scale, shake up, being mixed with mass concentration is the flat storing solution of Kui sulphur (IS Stock A, interior mark) of 543.7 μ g/mL, is stored in 4 ℃ of refrigerators stand-by.
(5) LC/MS/MS detects.
Wherein, method for separating liquid phase chromatography can be: chromatographic column (Ascentis, RP-Amide, 5 μ m, 50 * 2.1mm); Chromatogram flow phase: A is 5mM ammonium formate/5mM aqueous formic acid (55%), and B is methyl alcohol (45%); 40 ℃ of column temperatures; 6 ℃ of auto injection actuator temperatures; Wash pin liquid and be isopropanol water solution (1/1, v/v); 2 minutes analysis times, flow velocity 400 μ L/min, sample size 5 μ L.
Again, Mass Spectrometer Method method can be: scanning of the mass spectrum type is positive ion multiple-reaction monitoring (MRM); Ion gun: electric spray ion source (ESI); Atomization gas (NEB): 50psi; Air-flow: 9L/min; Capillary voltage (IS): 4500V; Ion source temperature (TEM): 350 ℃; Other parameters are as following table 5:
Table 5, Mass Spectrometer Method partial parameters:
| Compound | Q1(amu) | Q3(amu) | Dwell?time(msec) | Fragmentor(v) | CE(v) | Polarity(v) |
| Istain | 148 | 65.1 | 200 | 100 | 29 | Positive |
| Kui sulphur is put down (IS) | 384.1 | 253.1 | 200 | 100 | 21 | Positive |
(6) calculating of testing result:
A) above-mentioned typical curve sample is detected by above-mentioned LC/MS/MS detection method, and take peak area as y axle according to testing result, take drug concentration as x axle production standard curve;
B) equation of linear regression that passes through described typical curve with the plasma sample through the described such pre-treatment of step (1) through the value of above-mentioned LC/MS/MS detection method detection gained calculates 2 of correspondence, the concentration of 3-istain, related coefficient (the r of linear relationship between peak area ratio and concentration by the regression equation gained of 2,3-indolinedione
2) represent.
[method reliability compliance test]
Method of the present invention can be carried out reliability compliance test in the following manner.
1. minimum lower limit of quantitation sample preparation: get 2,3-indolinedione Quality Control storing solution (Stock B), be diluted to the standard solution of 0.4 μ g/mL with methanol solution.Get each 195 μ L of 6 parts of rat blank plasmas or Dog Plasma, add respectively above-mentioned 0.4 μ g/mL standard solution 5 μ L, prepare the minimum quantitative limit sample that 6 parts of mass concentrations are 10ng/mL, according to the albumen precipitation pre-treating method of above-mentioned plasma sample, process.
2. selectivity sample preparation: blank matrix sample (not adding interior mark): get blank rat or each 50 μ L of Dog Plasma of 6 parts of Different Individual, add 250 μ L methyl alcohol, vortex is centrifugal 5min(15000r/min after 60 seconds), get 100 μ L supernatants;
Zero-dose matrix sample (blank adds interior mark): get 1 part of blank rat or Dog Plasma 50 μ L, process according to the albumen precipitation pre-treating method of above-mentioned plasma sample.
3. the residual blank sample preparation of system: prepare blank matrix sample according to blank matrix sample (not adding interior mark) compound method, analyze after typical curve peak, investigation system is residual.
4. matrix effect sample and recovery sample preparation: the evaluation to matrix effect and the recovery, the sample by following preparation is investigated:
Quality-control sample preparation: get each 6 parts of the quality-control samples of three levels, process post analysis according to the albumen precipitation pre-treating method of above-mentioned plasma sample;
Recovery sample preparation: get the blank rat of three part of 195 μ L or Dog Plasma, use respectively 1000 μ L inner mark solution protein precipitations, vortex 60 seconds, the quality-control sample annex solution that adds respectively tri-levels of 5 μ L, vortex is centrifugal 5min(15000r/min after 60 seconds), get 100 μ L sample introduction analyses, 6 parts of each concentration level parallel processing;
Matrix effect sample preparation: the quality-control sample annex solution of getting tri-levels of 5 μ L adds 995 μ L methyl alcohol, vortex 60 seconds, gets 100 μ L sample introduction analyses, and each concentration level is got 6 parts;
The recovery of the comparative evaluation method by quality-control sample and recovery example pharmaceuticals peak area response; The matrix effect of the comparative evaluation method by recovery sample and matrix effect peak area response.Computing formula is as follows:
5. linearity: according in above-mentioned biological sample 2, the method for making of 3-istain quantitative test typical curve, in methodology, verify and prepare two parts of typical curve samples first, second and third day every day, albumen precipitation pre-treating method according to above-mentioned plasma sample is processed, and sample introduction is analyzed respectively at the beginning of methodology checking on the same day and while finishing.The same day, all typical curve sample result fitted to a typical curve simultaneously.
6. accuracy and the preparation of precision quality-control sample: take out Quality Control pond sample from refrigerator (80 ℃), process and sample introduction analysis according to the albumen precipitation pre-treating method of above-mentioned plasma sample.6 parts of each concentration level parallel processing.
7. stability sample preparation
Freeze-thaw stability: by the quality-control sample preparing (QC-Low, QC-Mid, QC-High, 6 parts of each concentration), deposit in-80 ℃ of refrigerators and take out after 24h, naturally melt under room temperature, again deposit in-80 ℃ of refrigerator freezings after sample dissolves completely.Freeze-thaw so repeatedly, after melting for the 3rd time, processes sample introduction analysis according to the albumen precipitation pre-treating method of above-mentioned plasma sample;
Sample stability after processing: by the quality-control sample (QC-Low, QC-Mid, QC-High, 6 parts of each concentration) after processing the same day, sample introduction analysis again after 6 ℃ of placement 24h, is used the quantitative test of second day typical curve on automatic sampler bottle plate;
4 ℃ of (on ice) stability of sample: by the quality-control sample preparing the same day (QC-Low, QC-Mid, QC-High, 6 parts of each concentration), 4 ℃ (on ice) places after 4h, processes sample introduction analysis according to the albumen precipitation pre-treating method of above-mentioned plasma sample;
Long-time stability: will be stored in-80 ℃ of quality-control samples (QC-Low, QC-Mid, QC-High, 6 parts of each concentration) in refrigerator, sample introduction analysis after processing by biological sample pre-treating method.The quality-control sample preparation time is separated by 24 days with analysis minute;
Storing solution stability: again weigh and prepare (being separated by 24 days) determinand 2,3-indolinedione storing solution (Stock C).With methyl alcohol dilution, after dilution, 2,3-indolinedione concentration is 40ng/mL.
[the reliability demonstration result of the assay method of 2,3-indolinedione concentration in rat and Beagle dog plasma sample]
1. typical curve: prepare typical curve sample three day every day, 2 parts of each concentration level preparations.The same day, all typical curve samples fitted to a typical curve simultaneously, the relative error of the Linear Points on result standard curve (RE) and relative standard deviation (RSD) are in 20%, curve correlation coefficient r2 is all more than 0.995, as table 6 and table 7, in following form, following symbol represents respectively: n: for counting statistics value number (Number of values used to calculate statistics); Conc.: concentration (Concentration); SD: standard deviation (Standard Deviation); %RE: relative error (Relative Error); RSD: relative standard deviation (Relative Standard Deviation); NA: inapplicable/unavailable (Not Applicable/Not Available)
Table 6, typical curve analysis result (rat, concentration unit: ng/mL):
Table 7, typical curve analysis result (Beagle dog, concentration unit: ng/mL):
2. selectivity: as shown in Figure 1 and Figure 4, the chromatogram of the blank matrix sample of Different Individual, at determinand (2,3-istain) and internal standard compound (Kui sulphur flat) go out Feng Chu, do not have Interference Peaks maybe can affect quantitative Interference Peaks, interfering effects of drug peak <LLOQ is corresponding 20%, mark corresponding 5% in interior mark Interference Peaks <.
3. minimum lower limit of quantitation: minimum lower limit of quantitation (LLOQ the is 10ng/mL) sample to the blank plasma preparation of 6 parts of Different Individual is analyzed, and the relative error of result specimen in use (RE) and relative standard deviation (RSD) are all in 20%, as table 8.In rat and Beagle dog plasma, minimum lower limit of quantitation sample chromatogram figure respectively as shown in Figure 2 and Figure 5;
Table 8, minimum lower limit of quantitation analysis result:
4. precision and accuracy: carry out analysis result demonstration to quality-control sample (QC-Low, QC-Mid, QC-High, 6 parts of each concentration) every day, the relative error of quality-control sample (RE) and relative standard deviation (RSD) all in 20%, as table 9 and table 10,
Table 9, rat plasma sample precision and accuracy analysis result:
Table 10, Beagle dog plasma sample precision and accuracy analysis result:
5. the residual result of system: to analyzing at the blank sample of the highest quantitative limit (ULOQ is 5000ng/mL) post analysis.In blank sample, do not have Interference Peaks maybe can affect quantitative Interference Peaks, interfering effects of drug peak <LLOQ is corresponding 20%, mark corresponding 5% in interior mark Interference Peaks <.In rat and Beagle dog plasma, system residual sample chromatogram respectively as shown in Figure 3 and Figure 6.
6. matrix effect and the recovery: matrix effect and recovery result of calculation are as table 11 and table 12, and the recovery approaches 100%, have certain matrix effect,
Table 11, rat plasma sample recovery rate and matrix effect analysis result:
Table 12, Beagle dog plasma sample recovery rate and matrix effect analysis result:
7. stability sample analysis: result demonstration, the relative error of sample (RE) and relative standard deviation (RSD) are in 20%, and as table 13, it is stable this sample being described under the condition of storage of regulation and in the time,
Table 13, stability sample analysis result:
Further exemplify embodiment below to describe the present invention in detail.Should understand equally; following examples are only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention., those skilled in the art can do in suitable scope and be selected by explanation herein the parameter that following example is concrete is only also an example in OK range,, and does not really want to be defined in the below concrete numerical value of example.
(test material)
Medicine and reagent:
Istain reference substance: Shanghai Xinshengyuan Medicine Research Co., Ltd. provides, lot number is 090601;
Internal standard compound Kui sulphur is flat: Mei Dixipuya (Shanghai) biological medicine company limited provides;
Methyl alcohol (Burdick & Jackson, HPLC grade), acetonitrile (Burdick & Jackson, HPLC grade), formic acid (ACROS ORGANICS, HPLC grade), water is ultrapure water.
Experimental apparatus:
TGL-16 high speed tabletop refrigerated centrifuge You Xiangyi hydro-extractor factory produces.LC system is Agilent 1200 series, and mass spectrometer system is Agilent 6410B, and data collection and control system software is Masshunter(Anjelen Sci. & Tech. Inc).Miniature vortex mixer (XW-80A, Industrial Co., Ltd. of upper Nereid section); Micro-analytical balance (XP26, Mettler-Toledo Instrument (Shanghai) Co., Ltd.); Micro porous filtration ultrapure water instrument.
Embodiment 1
Determination of plasma concentration in the body of SD rat administration 2,3-indolinedione
1. experimental animal: Sprague Dawley (SD) rat, male and female half and half, body weight 208.6～238.5g, by Shanghai, western pul-Bi Kai animal used as test company limited provides, credit number SCXK(Shanghai) 2008-0016;
2. dosage regimen: take appropriate 2,3-indolinedione, be formulated into desired concn according to dosage and administration volume, gavage is prepared with 1.25% tragcanth aqueous solution, intravenously administrable is with 5%DMSO, 40%PEG400,55% physiological saline preparation.Fasting 10～14h before animals administer, freely drinks water.Rat vein administration: take 20mg/kg dosage through the administration of tail intravenous injection (administration volume is 2mL/kg); Rat oral gavage administration: with 20,50 and the dosage of 100mg/kg respectively gavage give 2,3-indolinedione (administration volume is for 5mL/kg).After giving before tested material (0h) and giving tested material 0.083,0.25,0.5,1,2,4,6,8 and 24h by taking blood from jugular vein 0.20mL, K2EDTA anti-freezing, blood sample collection is placed on ice, centrifugal separation plasma (centrifugal condition: 8000 turn/min, 6min, 4 ℃).Before the plasma analysis of collecting, deposit in-80 ℃;
3. plasma sample assay method: adopt above-mentioned LC/MS/MS method to measure after rat administration the concentration of 2,3-indolinedione in different time points blood plasma.
Rat vein is injected 20mg/kg2, and the drug plasma concentration of medicine 2,3-indolinedione is in Table 14 after 3-istain (n=6), and rat oral gavage gives 20,50 and 100mg/kg2, and after 3-istain, the blood concentration of medicine is in Table 15-17:
Table 14, the blood concentration (ng/mL) of rat vein administration 20mg/kg different time:
Table 15, the blood concentration (ng/mL) of rat oral gavage administration 20mg/kg different time:
Table 16, the blood concentration (ng/mL) of rat oral gavage administration 50mg/kg different time:
Table 17, the blood concentration (ng/mL) of rat oral gavage administration 100mg/kg different time:
Embodiment 2
Determination of plasma concentration in the body of Beagle dog administration 2,3-indolinedione
1. experimental animal: beasle dog, male and female half and half, body weight 7.80～9.60kg, is purchased from Beijing Marshall Biotechnology Co., Ltd, credit number SCXK(capital) 2010-0003;
2. dosage regimen: take appropriate istain, be formulated into desired concn according to dosage and administration volume, gavage is prepared with 1.25% tragcanth aqueous solution, intravenously administrable is with 5%DMSO, 40%PEG400,55% physiological saline preparation.Fasting 10～14h before animals administer, freely drinks water.Beasle dog intravenously administrable: inject and give istain (administration volume is 1.5mL/kg) through saphena by the dosage of 15mg/kg; Beasle dog single gastric infusion: with 15,30 and the dosage of 60mg/kg respectively gavage give istain (administration volume is for 2mL/kg); Seven days successive administrations of beasle dog: take 30mg/kg dosage respectively gavage give istain (administration volume be 2mL/kg, once a day).After giving before tested material (0h) and giving tested material 0.083,0.25,0.5,1,2,4,6,8 and 24h through taking blood from jugular vein 1mL, be placed in the test tube containing K2EDTA, blood sample collection is placed on ice, centrifugal separation plasma (turn/min of centrifugal condition: 3500rpm, 10min, 4 ℃).Before the plasma analysis of collecting, deposit in-80 ℃;
3. plasma sample assay method: adopt above-mentioned LC/MS/MS method to measure after rat administration the concentration of istain in different time points blood plasma.
The blood concentration of the medicine istain after the istain of beasle dog intravenous injection 15mg/kg is in Table 18; Beasle dog on an empty stomach with 15,30 and 60mg/kg dosage gavage give istain, the plasma concentration of medicine istain is in Table 19-22:
Table 18, the blood concentration (ng/mL) of beasle dog intravenously administrable 15mg/kg different time:
Table 19, the blood concentration (ng/mL) of beasle dog oral administration 15mg/kg different time:
Table 20, the blood concentration (ng/mL) of beasle dog oral administration 30mg/kg different time:
Table 21, the blood concentration (ng/mL) of beasle dog oral administration 60mg/kg different time:
Table 22, the blood concentration (ng/mL) of continuous seven days oral administration 2,3-indolinedione (30mg/kg) different times of beasle dog:
Claims (10)
1. an assay method for the concentration of 2,3-indolinedione in plasma sample, is characterized in that, described assay method comprises: plasma sample pre-treatment step:
At 0～4 ℃, for mixing animal, 1:5 gives the plasma sample and the internal standard compound methanol solution that after 2,3-indolinedione, gather by volume, and vortex is centrifuging and taking supernatant after 30～90 seconds; And
Detecting step plasma sample through pre-treatment being detected by liquid chromatograph mass spectrography, wherein
Liquid phase chromatogram condition comprises: chromatographic column is Ascentis, RP-Amide, 5 μ m, 50 * 2.1mm; Chromatogram flow phase A is 5mM ammonium formate/5mM aqueous formic acid, and Mobile phase B is methyl alcohol, and the percent by volume of mobile phase A and B is respectively 55% and 45%;
Mass spectrum condition comprises: scanning of the mass spectrum type is positive ion multiple-reaction monitoring; Ion gun is electric spray ion source; Atomization gas pressure is 50psi; Gas flow rate is 9L/min; Capillary voltage is 4500V; Ion source temperature is 350 ℃, inner mark method ration.
2. assay method according to claim 1, is characterized in that, in described plasma sample pre-treatment step, and the flat methanol solution of Kui sulphur that described internal standard compound methanol solution is 10ng/mL.
3. assay method according to claim 1 and 2, is characterized in that, in described plasma sample pre-treatment step, described centrifugal be the centrifugal 5min of speed with 15000r/min.
4. according to the assay method described in any one in claims 1 to 3, it is characterized in that, liquid phase chromatogram condition also comprises: column temperature is 40 ℃; Auto injection actuator temperature is 6 ℃; Washing pin liquid is isopropanol water solution, and wherein the volume ratio of isopropyl alcohol and water is 1:1; Be 2 minutes analysis time, and flow velocity is 400 μ L/min, and sample size is 5 μ L.
5. according to the assay method described in any one in claim 1 to 4, it is characterized in that, described mass spectrum condition also comprises: the ion pair for quantitative test is: 2,3-indolinedione: 148 → 65.1; Kui sulphur is flat: 384.1 → 253.1.
6. according to the assay method described in any one in claim 1 to 5, it is characterized in that, described plasma sample is mammiferous blood plasma.
7. assay method according to claim 6, is characterized in that, described plasma sample is that animal gives the fresh plasma gathering after 2,3-indolinedione sample.
8. assay method according to claim 6, it is characterized in that, described plasma sample is that animal gives 2, the plasma sample gathering after 3-istain is deposited in the deposit plasma sample of-80 ℃ of refrigerators, described plasma sample pre-treatment step first takes out described deposit plasma sample before being also included in and mixing with described internal standard compound methanol solution from-80 ℃ of refrigerators, after naturally melting at 0～4 ℃, whirlpool is 20～40 seconds.
9. according to the assay method described in any one in claim 1～8, it is characterized in that, the lowest detection of described assay method is limited to 10ng/mL.
10. according to the assay method described in any one in claim 1～9, it is characterized in that, the relative error of described assay method and relative standard deviation are all in 20%.
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