CN101419224B - Method for simultaneously determining mycophenolic acid ester, mycophenolic acid and metabolite thereof in human blood plasma - Google Patents
Method for simultaneously determining mycophenolic acid ester, mycophenolic acid and metabolite thereof in human blood plasma Download PDFInfo
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- CN101419224B CN101419224B CN2008102024005A CN200810202400A CN101419224B CN 101419224 B CN101419224 B CN 101419224B CN 2008102024005 A CN2008102024005 A CN 2008102024005A CN 200810202400 A CN200810202400 A CN 200810202400A CN 101419224 B CN101419224 B CN 101419224B
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Abstract
The invention belongs to the field of medical test, and relates to a method for analyzing and measuring in vivo drugs, in particular to a method for measuring mycophenolate mofetil, mycophenolic acid and metabolites thereof in human plasma simultaneously. After the pretreatment of a sample to be measured, the method makes use of the characteristic of strong fluorescence absorption of the mycophenolate mofetil and the mycophenolic acid under an alkaline condition, performs derivatization after the analysis of chromatographic column separation, and detects with a fluorescence detector. The method can greatly improve the sensitivity for detecting the mycophenolate mofetil and the mycophenolic acid. The method has the advantages of few samples, simple pretreatment, rapidness, sensitivity, short analysis cycle and low cost, no needs of expensive equipment or reagents, and is suitable for clinical routine monitoring and pharmacokinetic study.
Description
Technical field
The invention belongs to field of medical examination, relate to the analysis determining method of drug disposition, be specifically related to a kind of method of measuring mycophenolate in the human plasma, mycophenolic acid and metabolin thereof.
Background technology
Anti-repelling treatment be need carry out after the clinical organ transplant, novel anti-metabolism immunodepressant mycophenolate (Mycophenolate Mofetil, MMF, RS61443 extensively adopted at present; Trade name: Cellcept, MMF) carries out anti-repelling treatment.This medicine is that (Mycophenolic Acid, 2-ethyl ester pro-drug MPA) can significantly improve bioavilability in the body of MPA to mycophenolic acid.After MMF is oral; Be hydrolyzed rapidly in vivo and take off ester and be converted into active metabolite MPA, the latter combines with glucuronic acid then, and being converted into does not have active metabolic product glucoside acidifying thing (Mycophenolic acid glucuronide; MPAG), its main metabolic pathway is as shown in Figure 1.MPAG concentration in vivo is much higher than MPA, and can be hydrolyzed to MPA once more through the circulation of intestines liver, get in the body again to play a role, so MPAG can influence the interior pharmacokinetics of body of MPA.Prior art research shows that the bioavilability of MMF in the part patient is lower than 80%, even is lower than 50%, and incomplete bio-transformation possibly influence the pharmacokinetics of MPA equally.So, through measuring the concentration of three kinds of materials in the blood plasma simultaneously, and adjust dosage in view of the above, significant to the safety and the curative effect that guarantee medication.
The prior art relevant with the present invention has patent: ZL 200410066519.6, but the medicine scope that wherein disclosed technical scheme can be measured still dislikes not enough.Up to now, do not see the report of home and abroad as yet about the concentration of measuring MMF in the human plasma, MPA and MPAG simultaneously.
Summary of the invention
The objective of the invention is to overcome the not enough defective of prior art, provide a kind of easy, quick, sensitive, can measure mycophenolate in the human plasma (Mycophenolate Mofetil, MMF, RS61443 simultaneously; Trade name: Cellcept, MMF), (Mycophenolic Acid is MPA) with metabolic product glucoside acidifying thing (Mycophenolic acid glucuronide, MPAG) method of concentration for mycophenolic acid.Easy and simple to handle, quick, few, the low cost and other advantages of blood plasma consumption that this method has need not expensive equipment and reagent, is more suitable for routine clinical monitoring and pharmacokinetic studies.
The technical scheme of this method is: after the testing sample pre-service; Earlier detect MPAG with UV-detector; Utilize MMF and MPA that the characteristic of strong fluorescent absorption is arranged under alkali condition then,, detect with fluorescence detector analyzing chromatographic column separation back online derivatization (alkalization).This method can make the sensitivity that detects of MMF and MPA improve greatly.
This method may further comprise the steps:
1) sample pretreatment
Get testing sample, add methyl alcohol, acetonitrile or both mixed liquors, carry out albumen precipitation; Get the supernatant sample introduction after centrifugal;
2) sample separation
Adopt universal liquid-phase chromatographic column, its filler is octadecyl or octyl bonding phase silica gel, and the high performance liquid chromatogram system adopts universal ultraviolet and fluorescence detector and high-pressure pump, and moving phase adopts isocratic elution; Adopt ultraviolet and fluorescence detector to detect MPAG respectively respectively, the peak area of MMF and MPA is converted into three's concentration with the typical curve equation; Wherein chromatographic condition is, the HPLC system: day island proper Tianjin LC-10A of company high performance liquid chromatogram appearance (system comprises double pump, UV-detector and fluorescence detector); Chromatographic column: ZORBAX RX-C8 (250mm * 4.6mm, 5 μ m); Moving phase: adopt isocratic elution, methyl alcohol-0.1%TFA (60:40-50:50, V/V); Flow velocity: 1.0-1.2ml/min; Column temperature 40-45 ℃; Fluorescence exciting wavelength 342-344nm, emission wavelength 425-427nm.
3) sample post column derivatization
Adopt derivatization reagent behind an additional high-pressure pump and the threeway joint pin, and connect UV-detector and fluorescence detector, make the sample after the separation that fluorescence signal arranged with series system.
This method is used for the concentration of the human plasma of quantitative measurement simultaneously MMF, MPA and MPAG: at first quantitatively obtain plasma sample; Add quantitative protein precipitant; Mixing, centrifugal is got the supernatant sample introduction, detects MPAG through UV-detector; After carrying out derivatization with NaOH or KOH solution again, detect MMF and MPA with fluorescence detector.
In the said determination method, the protein precipitant of adding is methyl alcohol, acetonitrile or the two potpourri.
In the said determination method, measure MPAG with UV-detector and fluorescence detector respectively, the peak area of MMF and MPA is converted into MPAG with the typical curve equation, the concentration of MMF and MPA.In the said method, adopt universal liquid-phase chromatographic column, its filler is that octadecyl or octyl bonding phase silica gel are the liquid-phase chromatographic column of filler.The high performance liquid chromatogram system adopts universal ultraviolet and fluorescence detector and high-pressure pump, and moving phase adopts isocratic elution.
The inventive method quick and precisely, easy and simple to handle, highly sensitive, cost is low, is fit to routine clinical monitoring and pharmacokinetic studies.Have following advantage: under this method condition determination, human body endogenous material and drug combination commonly used be interference measurement not all; Blood plasma MMF, MPA and MPAG setting-out line property scope are respectively 0.04～1.00 μ g/ml, 0.1～40.0 μ g/ml and 10～150 μ g/ml; Measurement deviation all in ± 15%, in a few days and precision in the daytime (relative standard deviation is RSD) all less than 12%.
Description of drawings
Fig. 1 is the main metabolic pathway figure of mycophenolate,
Wherein, MMF: mycophenolate; MPA: mycophenolic acid; MPAG: glucuronic acid mycophenolic acid.
Fig. 2 is a chromatographic system device synoptic diagram.
Fig. 3 is the typical color spectrogram, wherein,
Volunteer's blank plasma chromatogram: (a) fluorescence (b) ultraviolet;
The volunteer takes the blood plasma chromatogram of MMF 750mg bid, prednisone and ciclosporin A: (c) fluorescence (d) ultraviolet;
1:MMF; 2: interior mark; 3:MPA; 4:MPAG.
Fig. 4 is the typical color spectrogram, wherein,
Do not take the renal transplant recipients blank plasma chromatogram of MMF: (a) fluorescence (b) ultraviolet;
Actual renal transplant recipients is taken the blood plasma chromatogram of MMF 750mg bid, prednisone and ciclosporin A: (c) fluorescence (d) ultraviolet;
1:MMF; 2: interior mark; 3:MPA; 4:MPAG.
Embodiment
Embodiment 1: volunteer's blood plasma MMF, MPA and MPAG measure
Chromatographic condition
The HPLC system: (system comprises the LC-10AD pump to day island proper Tianjin LC-10A of company high performance liquid chromatogram appearance; The LC-10AT pump, SPD-10A UV-detector, RF-10AXL fluorescence detector; The SIL-10A automatic sampler; CBM-10A system communication device, column oven, Class-LC10 Version 1.63 chromatographic work stations); Chromatographic column: Kromasil-C18 (150mm * 4.6mm, 5 μ m); Moving phase: adopt isocratic elution, methyl alcohol---0.1%TFA (45:55, V/V); Flow velocity: 1.2ml/min; 40 ℃ of column temperatures; Fluorescence exciting wavelength 342nm, emission wavelength 425nm.
The plasma sample pre-service
The accurate 100 μ l blood plasma of drawing are put in the 1.5ml centrifuge tube, add the acetonitrile solution 200 μ l of mark Propafenone 150 μ g/ml in containing, vortex vibration 30s, and centrifugal 10min (20,627 * g, 4 ℃) gets supernatant 20 μ l sample introductions, and internal standard method is with peak area quantification.
Specificity
Get 8 volunteers' blank plasma, measure, do not find that endogenous material has interference to the said determination component according to above-mentioned sample pretreatment and assay method.In addition, common drug combination paracetamol, ACV, atenolol, imuran, Carvedilol, Ciprofloxacin, Clonazepam, Clozapine, ciclosporin A, diazepam, melbine, Doxazosin, FCV, fenofibrate, frusemide, GCV, Glipizide, Hydrochioro, brufen, Indomethacin, Irbesartan, Losartan, Metoclopramide, metoprolol, Ofloxacin, phenacetin, metacortandracin, Ribavirin, sirolimus, tacrolimus, Telmisartan, Valaciclovir, valganciclovir and Valsartan etc. do not disturb measuring yet.MMF, MPA, MPAG and interior target typical case chromatographic retention are respectively 6.5,15.7,4.9 and 10.8min, and whole stratographic analysis process time is 17.5min.
Linear test
Get blank plasma, add an amount of MMF, MPA and MPAG standard reserving solution are mixed with and contain MMF (0.040,0.125,0.250 respectively; 0.500,0.750,1.000 μ g/ml), MPA (0.1,0.5; 5.0,10.0,20.0,40.00 μ g/ml) and MPAG (10,20; 50,75,100,150 μ g/ml) blood plasma standard items, by " the plasma sample pre-service " the method operation.Internal standard method is made weighting (1/C with component to be measured and interior target peak area ratio (A) and concentration of component to be measured (C)
2) linear regression, three's typical curve regression equation is respectively: MMFC=0.2355A+0.0124, r=0.9996; MPA C=0.2004A+0.2034, r=0.9991; MPAGC=505.8589 A+1.6839, r=0.9996.
Accuracy and precision
Get blank plasma, add an amount of MMF, MPA and MPAG standard reserving solution are mixed with and contain MMF (0.04 respectively; 0.10,0.40,0.80 μ g/ml), MPA (0.10; 0.25,15.00,30.00 μ g/ml) and MPAG (10,25; 60,120 μ g/ml) blood plasma quality-control product, by " the plasma sample pre-service " the method operation, investigate in a few days and precision in the daytime and accuracy.Calculate its measured concentration according to equation of linear regression, calculate actual measurement mean value, deviation and the relative standard deviation (RSD) of every kind of concentration, wherein (C actual measurement-C is theoretical)/C theory * 100% is a deviation.
Table 1 has shown MMF in the blood sample, MPA and MPAG in a few days, day to day precision and accuracy.The result show three kinds of determinands in a few days with day to day precision all less than 11%, deviation is less than 15%.
Table 1
Embodiment 2: renal transplant recipients blood plasma MMF, MPA and MPAG measure
Chromatographic condition
The HPLC system: (system comprises the LC-10AD pump to day island proper Tianjin LC-10A of company high performance liquid chromatogram appearance; The LC-10AT pump, SPD-10A UV-detector, RF-10AXL fluorescence detector; The SIL-10A automatic sampler; CBM-10A system communication device, column oven, Class-LC10 Version 1.63 chromatographic work stations); Chromatographic column: ZORBAX RX-C8 (250mm * 4.6mm, 5 μ m); Moving phase: adopt isocratic elution, methyl alcohol---0.1%TFA (52:48, V/V); Flow velocity: 1.2ml/min; 45 ℃ of column temperatures; Fluorescence exciting wavelength 344nm, emission wavelength 427nm;
The plasma sample pre-service
The accurate 100 μ l blood plasma of drawing are put in the 1.5ml centrifuge tube, add the methanol solution 200 μ l of mark Propafenone 150 μ g/ml in containing, vortex vibration 30s, and centrifugal 10min (20,627 * g, 4 ℃) gets supernatant 20 μ l sample introductions, and internal standard method is with peak area quantification.
Specificity
Get 8 blank plasmas of not taking the renal transplant recipients of MMF, carry out the specificity evaluation, do not find that endogenous material and common drug have interference to the said determination component according to method among the embodiment 1.MMF, MPA, MPAG and interior target typical case chromatographic retention are respectively 6.6,15.6,4.9 and 10.7min, and whole stratographic analysis process time is 17.5min.
Linear test
Get blank plasma, add an amount of MMF, MPA and MPAG standard reserving solution are mixed with and contain MMF (0.040,0.125,0.250 respectively; 0.500,0.750,1.000 μ g/ml), MPA (0.1,0.5; 5.0,10.0,20.0,40.00 μ g/ml) and MPAG (10,20; 50,75,100,150 μ g/ml) blood plasma standard items, by " the plasma sample pre-service " the method operation.Internal standard method is made weighting (1/C with component to be measured and interior target peak area ratio (A) and concentration of component to be measured (C)
2) linear regression, three's typical curve regression equation is respectively: MMFC=0.2563A-0.0018, r=0.9997; MPA C=0.2181A+0.0050, r=0.9993; MPAGC=513.7937A+2.4051, r=0.9996.
Accuracy and precision
Get blank plasma, add an amount of MMF, MPA and MPAG standard reserving solution are mixed with and contain MMF (0.04 respectively; 0.10,0.40,0.80 μ g/ml), MPA (0.10; 0.25,15.00,30.00 μ g/ml) and MPAG (10,25; 60,120 μ g/ml) blood plasma quality-control product, by " the plasma sample pre-service " the method operation, investigate in a few days and precision in the daytime and accuracy.Calculate its measured concentration according to equation of linear regression, calculate actual measurement mean value, deviation and the relative standard deviation (RSD) of every kind of concentration, wherein (C actual measurement-C is theoretical)/C theory * 100% is a deviation.
Table 2 has shown MMF in the blood sample, MPA and MPAG in a few days, day to day precision and accuracy.The result show three kinds of determinands in a few days with day to day precision all less than 10%, deviation is less than 12%.
Table 2
Claims (3)
1. method of measuring mycophenolate in the human plasma, mycophenolic acid and metabolin thereof simultaneously; After it is characterized in that the testing sample pre-service; Analyzing chromatographic column separation back derivatization, detect with fluorescence detector then, said metabolin is mycophenolic acid glucoside acidifying thing MPAG; This method may further comprise the steps:
1) sample pretreatment
Get testing sample, add methyl alcohol, acetonitrile or both mixed liquors, carry out albumen precipitation; Get the supernatant sample introduction after centrifugal;
2) sample separation
Adopt universal liquid-phase chromatographic column, its filler is octadecyl or octyl bonding phase silica gel, and the high performance liquid chromatogram system adopts universal ultraviolet and fluorescence detector and high-pressure pump, and moving phase adopts isocratic elution; Adopt ultraviolet and fluorescence detector to detect metabolic product glucoside acidifying thing respectively respectively, the peak area of mycophenolate and mycophenolic acid is converted into three's concentration with the typical curve equation;
Chromatographic condition is the HPLC system: LC-10A high performance liquid chromatogram appearance; Chromatographic column: ZORBAX RX-C8250mm * 4.6mm, 5 μ m; Moving phase: adopt isocratic elution, methyl alcohol-0.1%TFA, both volume ratios are 60: 40-50: 50; Flow velocity: 1.0-1.2ml/min; Column temperature 40-45 ℃; Fluorescence exciting wavelength 342-344nm, emission wavelength 425-427nm;
3) sample post column derivatization
Adopt derivatization reagent behind an additional high-pressure pump and the threeway joint pin, and connect UV-detector and fluorescence detector, make the sample after the separation that fluorescence signal arranged with series system.
2. the method for measuring mycophenolate in the human plasma, mycophenolic acid and metabolin thereof simultaneously according to claim 1, the testing sample that it is characterized in that described step 1) is a human plasma.
3. method according to claim 1 is characterized in that in the described chromatographic condition, the HPLC system comprises double pump, UV-detector and fluorescence detector.
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| WO2005003175A2 (en) * | 2003-06-13 | 2005-01-13 | Biogen Idec Ma Inc. | Aglycosyl anti-cd154 (cd40 ligand) antibodies and uses thereof |
| CN1619304A (en) * | 2004-09-20 | 2005-05-25 | 复旦大学 | Method of determining mycophenolic acid in human blood plasma and its metabolite |
| WO2006058008A1 (en) * | 2004-11-23 | 2006-06-01 | Celgene Corporation | Methods and compositions using immunomodulatory compounds for treatment and management of central nervous system injury |
| CN101006986A (en) * | 2007-01-12 | 2007-08-01 | 杭州华东医药集团生物工程研究所有限公司 | Medicinal composition containing mycophonolate mofetil and its preparation method |
| CN101129380A (en) * | 2007-08-16 | 2008-02-27 | 江苏信孚药业有限公司 | Mycophenolate mofetil dispersible tablet and method of preparing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997029131A1 (en) * | 1996-02-09 | 1997-08-14 | Basf Aktiengesellschaft | HUMAN ANTIBODIES THAT BIND HUMAN TNF$g(a) |
| WO2005003175A2 (en) * | 2003-06-13 | 2005-01-13 | Biogen Idec Ma Inc. | Aglycosyl anti-cd154 (cd40 ligand) antibodies and uses thereof |
| CN1619304A (en) * | 2004-09-20 | 2005-05-25 | 复旦大学 | Method of determining mycophenolic acid in human blood plasma and its metabolite |
| WO2006058008A1 (en) * | 2004-11-23 | 2006-06-01 | Celgene Corporation | Methods and compositions using immunomodulatory compounds for treatment and management of central nervous system injury |
| CN101006986A (en) * | 2007-01-12 | 2007-08-01 | 杭州华东医药集团生物工程研究所有限公司 | Medicinal composition containing mycophonolate mofetil and its preparation method |
| CN101129380A (en) * | 2007-08-16 | 2008-02-27 | 江苏信孚药业有限公司 | Mycophenolate mofetil dispersible tablet and method of preparing the same |
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