CN102375040B - Method for determining content of simimtecan and chimmitecan in human or animal whole blood - Google Patents
Method for determining content of simimtecan and chimmitecan in human or animal whole blood Download PDFInfo
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- CN102375040B CN102375040B CN201010260758.0A CN201010260758A CN102375040B CN 102375040 B CN102375040 B CN 102375040B CN 201010260758 A CN201010260758 A CN 201010260758A CN 102375040 B CN102375040 B CN 102375040B
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Abstract
The invention provides a method for determining the content of simimtecan and chimmitecan in human or animal whole blood. The method comprises the steps of pretreating human or animal whole blood, carrying out separation on blood plasma and separating and detecting simimtecan and chimmitecan in the blood plasma by using the method of separation and detection; the invention is characterized in that the human or animal whole blood contacts with one carboxylesterase inhibitor or more carboxylesterase inhibitors in the process of pretreatment. According to the invention, the carboxylesterase inhibitors are utilized to block in vitro hydrolysis of simimtecan into chimmitecan, which guarantees that the result of in vivo exposure level inspection on the prodrug of simimtecan and the metabolite chimmitecan and the result of efficiency of conversion of the prodrug into the metabolite are reliable.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, relate to a kind of bright method of replacing health and lucky miaow to replace Kang Hanliang of happiness of measuring.More specifically, relate in a kind of human or animal's of mensuration whole blood, like bright for health and lucky miaow the method for Kang Hanliang.
Background technology
Liking bright is the camptothecin derivant of newly developing for health (9-allyl-10-(4-piperidinyl piperidine) formyloxy camptothecine), is used for the treatment of malignant tumour.This compound can be hydrolyzed into active metabolite-Ji miaow through carboxy-lesterase in vivo and produce antineoplastic curative effect for health (reaction equation 1).Carry out clinical before and in CLINICAL PHARMACOKINETIS STUDY ON, need to be to the happiness in animal and human's body plasma sample brightly for health and lucky miaow, for Kang Jinhang quantitative test, investigate exposure level in its body.
According to Literature Consult result, in existing analytical approach, no matter in processing, like the animal blood samples such as the bright rat for health, mouse, or when the bright human blood sample for health is liked in processing, all do not take the method for any blocking-up Carboxylesterase Activity.Wherein, some bibliographical informations adopts after blood sampling immediately centrifugal separation plasma and low temperature preserves until the method for analyzing
[1-7], some bibliographical information adopts whole blood to gather rearmounted method of preserving on ice
[2,4,7], some document is not mentioned related measure when gathering whole blood sample
[8-11].
Like the bright health of replacing extremely unstable in rat plasma, even if also can be generated lucky miaow for health by the carboxy-lesterase hydrolysis in blood plasma in vitro.If inhibition is timely and effectively liked the bright Kang Jixu of replacing and is hydrolyzed to lucky miaow for health when zoopery is taken a blood sample, can cause the bright interior exposure level miaow on the low side and lucky of health body that replaces of happiness to replace the higher illusion of exposure level in health body, cause the inaccuracy of experimental result.
The present invention is intended to for exposure level in health body and the bright ubiquitous false positive results of conversion ratio that replaces health to be converted into active metabolite of prodrug happiness, propose rational solution for the lucky miaow of current camptothecin cancer therapy drug: in analytical approach, adopt optionally carboxy-lesterase inhibitor-BNPP, in vitro the carboxy-lesterase in whole blood is suppressed, make the bright Kang Buzai of replacing of prodrug happiness continue to be in vitro converted into lucky miaow for health, thereby effectively guaranteed the accuracy of analytical approach.
List of references:
1.Escoriaza?J，Aldaz?A，Castellanos?C，Calvo?E，Giráldez?J.(2000)Simple?and?rapid?determination?of?irinotecan?and?its?metabolite?SN-38?in?plasma?by?high-performance?liquid-chromatography：application?to?clinical?pharmacokinetic?studies.J.Chromatogr.B，740：159-168.
2.Owens?TS，Dodds?H，Fricke?K，Hanna?SK，Crews?KR.(2003)High-performance?liquid?chromatographic?assay?with?fluorescence?detection?for?the?simultaneous?measurement?of?carboxylate?and?lactone?forms?of?irinotecan?and?three?metabolites?in?human?plasma.J.Chromatogr.B，788：65-74.
3.Escoriaza?J，Aldaz?A，Castellanos?C，Calvo?E，Giráldez?J.(2000)Simple?and?rapid?determination?of?irinotecan?and?its?metabolite?SN-38?in?plasma?by?high-performance?liquid-chromatography：application?to?clinical?pharmacokinetic?studies.J.Chromatogr.B，740：159-168.
4.de?Jong?FA，Mathijssen?RH，de?Bruijn?P，Loos?WJ，Verweij?J，Sparreboom?A.(2003)Determination?of?irinotecan?(CPT-11)and?SN-38?in?human?whole?blood?and?red?blood?cells?by?liquid?chromatography?with?fluorescence?detection.J.Chromatogr.B，795：383-388.
5.Poujol?S，Pinguet?F，Malosse?F，Astre?C，Ychou?M，Culine?S，Bressolle?F.(2003)Sensitive?HPLC-fluorescence?method?for?irinotecan?and?four?major?metabolites?in?human?plasma?and?saliva：application?to?pharmacokinetic?studies.Clin.Chem.，49：1900-1908.
6.Yang?X，Hu?Z，Chan?SY，Goh?BC，Duan?W，Chan?E，Zhou?S.(2005)Simultaneous?determination?of?the?lactone?and?carboxylate?forms?of?irinotecan?(CPT-11)and?its?active?metabolite?SN-38?by?high-performance?liquid?chromatography：application?to?plasma?pharmacokinetic?studies?in?the?rat.J.Chromatogr.B，821：221-228.
7.Khan?S，Ahmad?A，Guo?W，Wang?YF，Abu-Qare?A，Ahmad?I.(2005)A?simple?and?sensitive?LC/MS/MS?assay?for?7-ethyl-10-hydroxycamptothecin?(SN-38)in?mouse?plasma?and?tissues：application?to?pharmacokinetic?study?of?liposome?entrapped?SN-38(LE-SN38).J.Pharm.Biomed.Anal.，37：135-142.
8.Loos?WJ，de?Bruijn?P，Verweij?J，Sparreboom?A.(2000)Determination?of?camptothecin?analogs?in?biological?matrices?by?high-performance?liquid?chromatography.Anticancer?Drugs，11：315-324.
9.Vali?AM，Shafaghi?B，Dadashzadeh?S.(2005)Simple?and?sensitive?high?performance?liquid?chromatographic?method?for?the?simultaneous?quantitation?ofthe?lactone?and?carboxylate?forms?of?topotecan?in?human?plasma.J.Chromatogr.B，818：205-212.
10.D′Esposito?F，Tattam?BN，Ramzan?I，Murray?M.(2008)A?liquid?chromatography/electrospray?ionization?mass?spectrometry?(LC-MS/MS)assay?for?the?determination?of?irinotecan?(CPT-11)and?its?two?major?metabolites?in?human?liver?microsomal?incubations?and?human?plasma?samples.J.Chromatogr.B，875：522-530.
11.Bardin?S，Guo?W，Johnson?JL，Khan?S，Ahmad?A，Duggan?JX，Ayoub?J，Ahmad?I.(2005)Liquid?chromatographic-tandem?mass?spectrometric?assay?for?the?simultaneous?quantification?of?Camptosar?and?its?metabolite?SN-38?in?mouse?plasma?and?tissues.J.Chromatogr.A，1073：249-255.
12.Wadkins?RM，Hyatt?JL，Yoon?KJ，Morton?CL，Lee?RE，Damodaran?K，Beroza?P，Danks?MK，Potter?PM.(2004)Discovery?of?novel?selective?inhibitors?of?human?intestinal?carboxylesterase?for?the?amelioration?of?irinotecan-induced?diarrhea：synthesis，quantitative?structure-activity?relationship?analysis，and?biological?activity.Mol.Pharmacol.，65：1336-1343.
Summary of the invention
For improve in animal and human's body whole blood/plasma sample, like bright for health and lucky miaow the accuracy for Kangding component analysis, by adopting, carboxy-lesterase inhibitor blocking-up happiness is bright to be continued in vitro hydrolysis for health and generates lucky miaow for health in the present invention, thereby has guaranteed that prodrug happiness is bright for health with the lucky miaow of metabolic product is investigated for exposure level in health body and prodrug is converted into the reliability of the transformation efficiency result of metabolic product.This technical scheme is equally applicable to the plasma sample analysis of other camptothecine compounds (one-tenth ester prodrugs).
Therefore, the object of this invention is to provide in a kind of human or animal's of mensuration whole blood, like bright for health and lucky miaow the method for Kang Hanliang.
In a kind of human or animal's of mensuration whole blood that object according to the present invention provides, like the bright method of replacing health and lucky miaow to replace Kang Hanliang, the method comprises carries out after pre-service human or animal's whole blood, separated plasma, adopt again method for separating and detecting to liking bright health and the lucky miaow of replacing in blood plasma for Kang Jinhang separation and detection, wherein, human or animal's whole blood is being carried out in preprocessing process, human or animal's whole blood is being contacted with one or more carboxy-lesterase inhibitor.
Even due in vitro, happiness in human or animal's whole blood is bright also can be generated lucky miaow for health by carboxy-lesterase hydrolysis wherein for health.Therefore,, for the bright hydrolysis for health of inhibition happiness more early, the human or animal's of collection whole blood should contact with one or more carboxy-lesterase inhibitor as early as possible.For example, one or more carboxy-lesterase inhibitor can preexist in heparin tube; Or in the backward human or animal's that takes a blood sample whole blood, add rapidly carboxy-lesterase inhibitor.
Preferably, described one or more carboxy-lesterase inhibitor can preexist in heparin tube.Thus, the human or animal's of collection whole blood can contact with these one or more carboxy-lesterase inhibitor in time, thereby more effectively suppress the bright health of replacing of happiness, is hydrolyzed to lucky miaow for health.
For the human or animal's of collection whole blood is contacted fully with one or more carboxy-lesterase inhibitor, preferably by these one or more carboxy-lesterase inhibitor, the form with powder is added in heparin tube, and shakes up rapidly when contact.
Described carboxy-lesterase inhibitor can be two (p-nitrophenyl) phosphates (BNPP, bis-p-nitrophenyl-phosphate), thenoyltrifluoroacetone (thenoyltrifluoroacetone) or dibenzoyl (benzil).
Preferably, described carboxy-lesterase inhibitor is BNPP.BNPP is a species specific carboxy-lesterase inhibitor, and it is nonreversibility to the inhibition of carboxy-lesterase.Wadkins etc. are carrying out when people's small intestine carboxy-lesterase inhibitor synthesizes with activity identification usining BNPP as positive control compound
[12].The inventor investigates carboxy-lesterase inhibiting effect in the rat plasma of BNPP, and its Carboxylesterase Activity inhibiting rate can reach more than 98%, therefore selects BNPP as the carboxy-lesterase inhibitor of rat blood sample.
(for example, the ratio of ml BNPP): mg is 1: 5～1: 10, is preferably 1: 5, can suppress like this to like the bright hydrolysis for health substantially completely for described human or animal's whole blood and described carboxy-lesterase inhibitor.
In order to prevent human or animal's the rapid blood coagulation of whole blood, the inwall of described heparin tube can be coated with anticoagulant, for example liquaemin.
In described assay method, the process of separated plasma comprises and (for example, 3000rpm) obtains blood plasma by the whole blood of the human or animal after fully contacting with one or more carboxy-lesterase inhibitor is centrifugal.Further with acetonitrile, blood plasma is carried out albumen precipitation and centrifugally (for example, 13000rpm), obtains supernatant for separating of detection.
Described method for separating and detecting can adopt well known by persons skilled in the art can reaching liking bright any method of replacing health and lucky miaow to replace Kang Jinhang separation and detection object.
Preferably, described separation method is high performance liquid chromatography.
Described high performance liquid chromatography is used highly effective liquid phase chromatographic system, and actual conditions those skilled in the art of this system just can obtain by simple experiment.Also can be referring to document 1-11.
Preferably, the condition of described highly effective liquid phase chromatographic system is:
Chromatographic column: C
18(50mm * 2.1mm; 5 μ m);
Column temperature: room temperature;
Mobile phase: solvent orange 2 A: CH
3cN-H
2o (v/v, 1: 99; 0.4 ‰ HCOOH; 0.05 ‰ HCOONH
4); Solvent B:CH
3cN-H
2o (v/v, 99: 1; 0.4 ‰ HCOOH; 0.05 ‰ HCOONH
4);
Condition of gradient elution: 0～0.5min is with 4% solvent B wash-out, and 0.5～2min solvent B is at the uniform velocity increased to 100%, 2～3min with 100% solvent B wash-out from 4%, and 3～4min carries out column equilibration with 4% solvent B;
Flow velocity: 0.40mL/min;
Sample size: 5 μ L.
Described detection method is (series connection) Mass Spectrometer Method, is specially: adopt positive ion electron spray ionisation pattern (ESI+), under the collision energy of MRM pattern and 49V, detect bright m/z 599 → 124 ion pairs for health of happiness; Under the collision energy of 35V, detect lucky miaow for m/z 405 → 361 ion pairs of health.
It will be understood by those skilled in the art that for the bright mensuration of replacing health and lucky miaow to replace Kang Hanliang of happiness, can adopt separation or detection method under different condition to carry out, as long as these methods are applicable to happiness, bright health or the lucky miaow of replacing replaces health.
Certainly, for convenient, preferably use separation or detection method under the same terms, can reach like this object of Fast Measurement.
In mensuration of the present invention human or animal whole blood, like that bright the method for Kang Hanliang is specially for health and lucky miaow: the whole blood that adds human or animal in inwall is in advance coated with liquaemin and adds the heparin tube of carboxy-lesterase inhibitor, shake up gently, centrifuging obtains blood plasma, in this blood plasma, add acetonitrile to carry out albumen precipitation, obtain supernatant after centrifugal; Described supernatant is carried out in LC-MS instrument to separation and detection.
It will be understood by those skilled in the art that in assay, also need preparation standard curve.
In mensuration human or animal whole blood of the present invention, like bright health and the lucky miaow of replacing in the method for Kang Hanliang, when carrying out the standard curve making of quantitative test, take preprocess method preparation happiness same as described above bright for health standard plasma solutions: in the heparin tube that has in advance carboxy-lesterase inhibitor, to add blank plasma or (for example add a certain amount of carboxy-lesterase inhibitor in blank plasma, BNPP) after powder, add again happiness bright for health and lucky miaow for health standard items be mixed with containing finite concentration happiness bright for health and lucky miaow the standard plasma solutions for health, and dilute with the blank plasma containing carboxy-lesterase inhibitor, be mixed with bright health and the lucky miaow of replacing of happiness of a series of concentration for health standard plasma solutions.Then to this happiness is bright, for health and lucky miaow, in health standard plasma solutions, add acetonitrile to carry out albumen precipitation, the supernatant obtaining after centrifugal is for LC-MS separation and detection.
In assay method of the present invention, adopt specific carboxy-lesterase inhibitor (for example, BNPP) carboxy-lesterase in blood plasma to be suppressed effectively, thereby blocking-up happiness is bright, for health continuing in vitro, be hydrolyzed into lucky miaow for health.Thereby guaranteed that prodrug happiness is bright for health with the lucky miaow of metabolic product is investigated for exposure level in health body and prodrug is converted into the reliability of the transformation efficiency result of metabolic product.
Be appreciated that assay method of the present invention can be applied in the assay of any compound that can be hydrolyzed by carboxy-lesterase.For example, as long as compound and the carboxy-lesterase measured are present in medium (, blood plasma or serum) simultaneously.
Accompanying drawing explanation
Fig. 1 be rat vein give (dosage is 2.0mg/kg) like bright for after health, adopt the method for prior art measure happiness bright for health (left scale) and the lucky miaow of metabolic product the blood concentration-time plot for health (right scale).
Fig. 2 be rat (female) vein (i.v.) give (dosage is 3.75mg/kg, 7.5mg/kg, 15mg/kg) like bright for after health, adopt method of the present invention measure happiness bright for health and lucky miaow the blood concentration-time plot for health.
Fig. 3 adds carboxy-lesterase inhibitor whether on liking the bright impact for health blood plasma typical curve result in rat plasma under external room temperature different condition.(A) happiness that adds carboxy-lesterase inhibitor B NPP to obtain is bright for health blood plasma typical curve; (B) happiness that does not add carboxy-lesterase inhibitor B NPP to obtain is bright for health blood plasma typical curve.
Fig. 4 LC-MS detection method together measure in rat plasma, like bright for health and lucky miaow the chromatogram for health.A, blank rat plasma sample; B, has added in blank rat plasma sample and has liked the bright standard items that replace health and lucky miaow to replace health; C, plasma sample after rat vein administration.
Embodiment
LC-MS system: the AcQuity that liquid phase-mass spectrometry analytic system (LC/MS/MS) is produced by U.S. Waters company
tMthe API4000 Qtrap mass spectrometer that UPLC series liquid chromatograph instrument and Applied Biosystems company produce forms, system works software is EMPOWER Pro (U.S.) and Analysis1.4.2 (U.S.), controls respectively liquid phase and mass spectrometer system.
HPLC level acetonitrile (CH
3cN), methyl alcohol (MeOH), formic acid (HCOOH) and ammonium formate (HCOONH
4) be the reagent that U.S. Merck company produces.Experimental water is prepared by Millipore Direct-Q.Other organic reagent is provided by China Medicine (Group) Shanghai Chemical Reagent Co.,, analyzes pure.
Experimental technique:
1, animal blood specimen collection: add rapidly carboxy-lesterase inhibitor B NPP (every 1mL blood approximately adds 5mg) after animal blood taking, shake up rear centrifuging and taking blood plasma, blood plasma is to be measured in-70 ℃ of preservations.
2, rat plasma sample pre-treatments: the plasma sample of-70 ℃ of freezing preservations (50 μ L) room temperature is thawed, add acid acetonitrile solution (containing 0.5% formic acid) the 150 μ L joltings containing the interior mark compound of 100ng/mL to extract (1600rpm, 5min), centrifugal (13,000rpm, 5min, 4 ℃) after, get supernatant for analyzing.
3, LC-MS analysis condition: chromatographic column: Agilent Eclipse Plus C
18(50mm * 2.1mm; 5 μ m, Agilent, USA); Column temperature: room temperature; Mobile phase: solvent orange 2 A: CH
3cN-H
2o (v/v, 1: 99; 0.4 ‰ HCOOH; 0.05 ‰ HCOONH
4); Solvent B:CH
3cN-H
2o (v/v, 99: 1; 0.4 ‰ HCOOH; 0.05 ‰ HCOONH
4); Condition of gradient elution: 0～0.5min is with 4% solvent B wash-out, and 0.5～2min solvent B is at the uniform velocity increased to 100%, 2～3min with 100% solvent B wash-out from 4%, and 3～4min carries out column equilibration with 4% solvent B; Flow velocity: 0.40mL/min; Sample size: 5 μ L.Mass Spectrometer Method adopts positive ion electron spray ionisation pattern (ESI+), under the collision energy of MRM pattern and 49V, detects happiness bright for health m/z 599 → 124 ion pairs; Under the collision energy of 35V, detect lucky miaow for m/z 405 → 361 ion pairs of health, mass spectrum running parameter is in Table 1.
The bright mass spectrum running parameter that replaces health and lucky miaow to replace health of table 1 application LC-MS technology Simultaneous Quantitative Analysis happiness
4, the preparation of typical curve: to liking the bright acetonitrile standard reserving solution (concentration is 100 μ g/mL) that replaces health and lucky miaow to replace health containing adding in the blank rat plasma (blood plasma (ml): BNPP (mg)=1: 5) of BNPP, be made into and be equivalent to for health and lucky miaow, replace the plasma sample that health concentration is 1000ng/mL containing happiness is bright, then by the blank rat plasma dilution that contains BNPP for 3 times of intervals, preparation happiness is bright replaces for health and lucky miaow the series of standards sample that health concentration range is 1.37～1000ng/mL.With after the method cleanup standard sample " experimental technique 2 " Suo Shu (50 μ L), then obtain sample detection response (ratio of test compound and interior mark chromatographic peak peak area) by the method for " experimental technique 3 ".The sign concentration (X, ng/mL) of above-mentioned standard blood sample of take is independent variable, take that to detect response (Y) be dependent variable, carries out linear regression, and take 1/X as weight coefficient by least square method, tries to achieve the regression equation of measuring effect and concentration.
Adopt bibliographical information
[2]method (that is: whole blood after gathering, preserve immediately by centrifugal separation plasma low temperature, in analytic process, sample is put in ice bath, and after blood sampling, do not block immediately Carboxylesterase Activity) measured vein like bright in rat plasma after health (2mg/kg), like bright for health and lucky miaow the concentration for health, resulting their blood concentration-time curve is shown in Fig. 1.Wherein, AUC
like bright for healthbe 170 ± 6ngh/mL, AUC
lucky miaow is for healthbe 105 ± 4ngh/mL, AUC
lucky miaow is for health/ AUC
like bright for healthratio be 61.8%.
The method that adds immediately carboxy-lesterase inhibitor B NPP blocking-up Carboxylesterase Activity after adopting blood sampling of the present invention, to rat (female) vein (i.v.), giving (dosage is 3.75mg/kg, 7.5mg/kg, 15mg/kg) likes bright while analyzing for the blood sample after health, like and brightly for health is external, continues to be converted into lucky miaow and obtained good inhibition for health, resulting happiness is bright sees Fig. 2 for health and lucky miaow for the blood concentration-time curve of health.AUC under three dosage
lucky miaow is for health/ AUC
like bright for healthratio be respectively 6.74% (3.75mg/kg dosage group), 4.51% (7.5mg/kg dosage group) and 3.74% (15mg/kg dosage group).
Under the external room temperature different condition of embodiment 2, in rat plasma, add carboxy-lesterase inhibitor whether on liking the bright health of replacing, to be converted into lucky miaow for the impact of health
Experimental technique:
1, sample preparation: to containing the blank rat plasma (blood plasma (ml): BNPP (mg)=1: 5) of BNPP or containing in the blank rat plasma of BNPP, add the bright acetonitrile standard reserving solution (concentration is 100 μ g/mL) for health of happiness, be made into be equivalent to containing happiness bright for health concentration, be the plasma sample of 333ng/mL and 37.1ng/mL.
2, rat plasma sample pre-treatments: with embodiment 1.
3, LC-MS analysis condition: with embodiment 1.
The results are shown in Table 2.
Under the external room temperature different condition of table 2, in rat plasma, add carboxy-lesterase inhibitor B NPP whether on liking the bright health of replacing, to be converted into lucky miaow for the impact of health
The result of table 2 shows, adds in advance after a certain amount of carboxy-lesterase inhibitor B NPP in heparin tube, likes brightly for health, to continue to be in vitro converted into lucky miaow and obtained completely suppressing for health, like bright for health without conversion, in sample, do not detect lucky miaow for health; And in heparin tube, do not add carboxy-lesterase inhibitor, liking the bright conversion ratio for health is 63.4-66.0%.
Under the external room temperature different condition of embodiment 3, in rat plasma, add carboxy-lesterase inhibitor whether on liking the bright impact for health blood plasma typical curve result
Experimental technique:
1, the preparation of typical curve: to the blank rat plasma (blood plasma (ml): BNPP (mg)=1: 5) containing BNPP with not containing adding the bright acetonitrile standard reserving solution (concentration is 100 μ g/mL) for health of happiness in the blank rat plasma of BNPP, be made into and be equivalent to containing the bright plasma sample that is 6000ng/mL for health concentration of happiness, then by the blank rat plasma dilution containing BNPP for 3 times of intervals, the bright series of standards sample that is 1.37～6000ng/mL for health concentration range of preparation happiness.
2, rat plasma sample pre-treatments: with embodiment 1.
3, LC-MS analysis condition: with embodiment 1.
In blood plasma, add the bright health typical curve that replaces of the latter made happiness of carboxy-lesterase inhibitor B NPP in the concentration range internal linear good (r=0.9941) of 1.37-6000ng/mL; In blood plasma, do not add the bright health typical curve that replaces of the latter made happiness of carboxy-lesterase inhibitor in same concentration range internal linear poor (r=0.9447), show to like the bright health of replacing unstable in standard curve making process, have certain conversion (Fig. 3).
Fig. 4 be LC-MS detection method together measure in rat plasma, like bright for health and lucky miaow the chromatogram for health.A, blank rat plasma sample; B, has added in blank rat plasma sample and has liked the bright standard items that replace health and lucky miaow to replace health; C, plasma sample after rat vein administration.This figure is the representative chromatogram that carries out above-mentioned 3 kinds of embodiment gained.
In sum, adopt and of the present invention human or animal's whole blood is being carried out in preprocessing process, human or animal's whole blood is contacted with one or more carboxy-lesterase inhibitor, one or more carboxy-lesterase inhibitor can preexist in heparin tube; Or in the backward human or animal's that takes a blood sample whole blood, add immediately after the method for carboxy-lesterase inhibitor blocking-up Carboxylesterase Activity, like and brightly for health, continue to be in vitro converted into lucky miaow and obtained completely suppressing for health, resulting happiness is bright replaces the blood concentration of health can reflect preferably its actual conditions in vivo for health and lucky miaow, has guaranteed that camptothecine compounds prodrug is converted into the reliability of the transformation efficiency result of metabolic product.
Claims (7)
- One kind measure in human or animal's whole blood, like bright for health and lucky miaow the method for Kang Hanliang, the method comprises carries out after pre-service human or animal's whole blood, separated plasma, adopt again method for separating and detecting to liking bright health and the lucky miaow of replacing in blood plasma for Kang Jinhang separation and detection, it is characterized in that, human or animal's whole blood is being carried out in preprocessing process, human or animal's whole blood is being contacted with one or more carboxy-lesterase inhibitor; Described one or more carboxy-lesterase inhibitor preexist in heparin tube; And the ratio of the ml:mg of human or animal's whole blood and described carboxy-lesterase inhibitor is 1:5～1:10.
- 2. in mensuration human or animal whole blood according to claim 1, like bright health and the lucky miaow of replacing for the method for Kang Hanliang, it is characterized in that, described carboxy-lesterase inhibitor is two (p-nitrophenyl) phosphates, thenoyltrifluoroacetone or dibenzoyl.
- 3. in mensuration human or animal whole blood according to claim 1, like bright health and the lucky miaow of replacing for the method for Kang Hanliang, it is characterized in that, the ratio of the ml:mg of human or animal's whole blood and described carboxy-lesterase inhibitor is 1:5.
- 4. in mensuration human or animal whole blood according to claim 1, like bright health and the lucky miaow of replacing for the method for Kang Hanliang, it is characterized in that, the inwall of described heparin tube is coated with liquaemin.
- 5. in mensuration human or animal whole blood according to claim 1, like bright health and the lucky miaow of replacing for the method for Kang Hanliang, it is characterized in that, separated plasma process comprises the centrifugal blood plasma that obtains of whole blood of the human or animal after contacting with one or more carboxy-lesterase inhibitor.
- 6. in mensuration human or animal whole blood according to claim 1, like bright health and the lucky miaow of replacing for the method for Kang Hanliang, it is characterized in that, described separation method is high performance liquid chromatography; Detection method used is Mass Spectrometer Method.
- One kind measure in human or animal's whole blood, like bright for health and lucky miaow the method for Kang Hanliang, the method is specially: the whole blood that adds human or animal in inwall is in advance coated with liquaemin and adds the heparin tube of carboxy-lesterase inhibitor, shake up, centrifuging obtains blood plasma, in this blood plasma, add acetonitrile to carry out albumen precipitation, obtain supernatant after centrifugal; Described supernatant is carried out in LC-MS instrument to separation and detection, wherein, the ratio of the ml:mg of human or animal's whole blood and described carboxy-lesterase inhibitor is 1:5～1:10.
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| CN110220757B (en) * | 2019-06-05 | 2021-08-24 | 浙江龙传生物医药科技有限公司 | Blood preservative for drug metabolism detection |
| CN110208411B (en) * | 2019-06-10 | 2021-12-24 | 浙江龙传生物医药科技有限公司 | Carboxylesterase inhibitor formulations for drug metabolism detection |
| CN110161159B (en) * | 2019-07-04 | 2020-09-22 | 杭州必益泰得医学科技有限公司 | Biological sample analysis method for oxcarbazepine bioequivalence test |
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| WO2003014069A1 (en) * | 2001-08-10 | 2003-02-20 | Pharmacia Italia Spa | Fluoro linkers and their use as linkers for enzyme-activated drug conjugates |
| CN1616460A (en) * | 2003-11-10 | 2005-05-18 | 中国科学院上海药物研究所 | Novel derivative of camptothecine, preparing method and use |
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| WO2003014069A1 (en) * | 2001-08-10 | 2003-02-20 | Pharmacia Italia Spa | Fluoro linkers and their use as linkers for enzyme-activated drug conjugates |
| CN1616460A (en) * | 2003-11-10 | 2005-05-18 | 中国科学院上海药物研究所 | Novel derivative of camptothecine, preparing method and use |
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| A liquid chromatography/electrospray ionization mass spectrometry (LC–MS/MS) assay for the determination of irinotecan (CPT-11) and its two major metabolites in human liver microsomal incubations and human plasma samples;Fabrizio D’Esposito et al;《Journal of Chromatography B》;20081115;第875卷(第2期);522-530页全文 * |
| Eiji Araki et al.Relationship between Development of Diarrhea and the Concentration of SN-38, an Active Metabolite of CPT-11, in the Intestine and the Blood Plasma of Athymic Mice Following Intraperitoneal Administration of CPT-11.《Jpn. J. Cancer Res.》.1993,第84卷697-702. |
| Fabrizio D’Esposito et al.A liquid chromatography/electrospray ionization mass spectrometry (LC–MS/MS) assay for the determination of irinotecan (CPT-11) and its two major metabolites in human liver microsomal incubations and human plasma samples.《Journal of Chromatography B》.2008,第875卷(第2期),522-530. |
| Relationship between Development of Diarrhea and the Concentration of SN-38, an Active Metabolite of CPT-11, in the Intestine and the Blood Plasma of Athymic Mice Following Intraperitoneal Administration of CPT-11;Eiji Araki et al;《Jpn. J. Cancer Res.》;19930630;第84卷;697页摘要及右栏第3段 * |
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