CN101419225B - Method for simultaneously determining mycophenolic acid ester, mycophenolic acid and metabolite thereof in human urine - Google Patents
Method for simultaneously determining mycophenolic acid ester, mycophenolic acid and metabolite thereof in human urine Download PDFInfo
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- CN101419225B CN101419225B CN200810202401A CN200810202401A CN101419225B CN 101419225 B CN101419225 B CN 101419225B CN 200810202401 A CN200810202401 A CN 200810202401A CN 200810202401 A CN200810202401 A CN 200810202401A CN 101419225 B CN101419225 B CN 101419225B
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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 urine 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, no needs of expensive equipment or reagents, short analysis cycle and low cost, 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 urine, mycophenolic acid and metabolin thereof simultaneously.
Background technology
Mycophenolate (Mycophenolate Mofetil, MMF, RS61443; Trade name: Cellcept, MMF) is a kind of novel anti-metabolism immunodepressant, is widely used in the anti-repelling treatment after the organ transplant clinically.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.Existing simultaneously a plurality of researchs show 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.In addition, because MMF, MPA and MPAG be in vivo by urine excretion, so, through measuring the concentration of three kinds of materials in the urine simultaneously, and adjust dosage in view of the above, significant to the safety and the curative effect of assurance medication.
Up to now, do not see the report of home and abroad as yet about the concentration of measuring MMF in the human urine, MPA and MPAG simultaneously.
Summary of the invention
The purpose of this invention is to provide a kind of easy, quick, sensitive, can measure (the Mycophenolate Mofetil of mycophenolate in the urine simultaneously; MMF), mycophenolic acid (Mycophenolic Acid; MPA) and glucoside acidifying thing (Mycophenolic acid glucuronide, MPAG) method of concentration.This method need not expensive equipment and reagent, has easy and simple to handle, quick, few, the low cost and other advantages of urine consumption, is fit to 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 obviously improve.
The inventive method may further comprise the steps:
1) sample pretreatment: get testing sample, add methyl alcohol, deionized water or both mixed liquors, carry out the urine sample dilution, 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;
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 urine MMF, MPA and MPAG setting-out line property scope are respectively 0.075～1.000 μ g/ml, 0.1～10.0 μ g/ml and 20～400 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 13%.
This method adopts derivatization reagent behind an additional high-pressure pump and the threeway joint pin, and connects UV-detector and fluorescence detector with series system, carries out online derivatization after the sample column, can make the sample after the separation that fluorescence signal is arranged.
This method is used for the urine of quantitative measurement simultaneously MMF, MPA and MPAG concentration; At first quantitatively obtain urine sample; Add quantitative urine thinning agent, mixing, centrifugal is got the supernatant sample introduction; Detect MPAG through UV-detector, carry out detecting MMF and MPA with fluorescence detector behind the derivatization with NaOH or KOH solution again.In the said determination method, the urine thinning agent of adding is methyl alcohol, deionized water 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.
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: C8 analytical column; Moving phase: adopt isocratic elution, methyl alcohol-0.1%TFA (50:50-60:40, V/V); Flow velocity: 1.0-1.2ml/min; Column temperature 40-45 ℃; Fluorescence exciting wavelength 342-344nm, emission wavelength 425-427nm.
The inventive method can be measured mycophenolate in the human urine, mycophenolic acid and metabolin thereof simultaneously, quick and precisely, easy and simple to handle, highly sensitive, cost is low, is fit to routine clinical monitoring and pharmacokinetic studies.Has following advantage: under this method condition determination; Human body endogenous material and drug combination commonly used be interference measurement not all; Urine MMF, MPA and MPAG setting-out line property scope are respectively 0.075～1.000 μ g/ml, 0.1～10.0 μ g/ml and 20～400 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 13%.
Description of drawings
Fig. 1 is the main metabolic pathway of mycophenolate,
Wherein, MMF: mycophenolate; MPA: mycophenolic acid; MPAG: glucuronic acid mycophenolic acid.
Fig. 2 is a chromatographic system device synoptic diagram.
Fig. 3: typical color spectrogram: the blank urine chromatogram of volunteer: (a) fluorescence (b) ultraviolet;
The volunteer takes the urine chromatogram of MMF 750mg bid, prednisone and ciclosporin A: (c) fluorescence, (d) ultraviolet;
1:MMF wherein; 2:MPA; 3:MPAG
Fig. 4; Typical color spectrogram: the blank urine chromatogram of renal transplant recipients of not taking MMF: (a) fluorescence (b) ultraviolet;
Actual renal transplant recipients is taken the urine chromatogram of MMF 750mg bid, prednisone and ciclosporin A: (c) fluorescence (d) ultraviolet;
1:MMF wherein; 2:MPA; 3:MPAG.
Embodiment
Embodiment 1: volunteer's urine sample 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 urine sample pre-service
The accurate 100 μ L urine samples of drawing are put in the 1.5mL centrifuge tube, add methanol solution 500 μ L dilution, vortex vibration 15s, and centrifugal 5min (20,627 * g, 4 ℃) gets supernatant 20 μ L sample introductions, and external standard method is with peak area quantification.
Specificity
Get 8 blank plasmas of not taking the renal transplant recipients of MMF of separate sources, 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 urine, add an amount of MMF, MPA and MPAG standard reserving solution are mixed with and contain MMF (0.075,0.125,0.250 respectively; 0.500,0.750,1.000 μ g/ml), MPA (0.1,0.5; 1.0,2.5,5.0,10.0 μ g/ml) and MPAG (20,50; 80,100,200,400 μ g/ml) urine standard items, by " the urine sample pre-service " the method operation.External standard method is done weighting (1/C2) linear regression with the peak area (A) and the concentration of component to be measured (C) of component to be measured, and three's typical curve regression equation is respectively: MMF C=7.9486 * 10
-6A-0.0034, r=0.9991; MPA8.5041 * 10
-6A+0.0086, r=0.9994; MPAG C=0.0013A+6.2899, r=0.9999
Accuracy and precision
Get blank urine, add an amount of MMF, MPA and MPAG standard reserving solution are mixed with and contain MMF (0.075 respectively; 0.150,0.400,0.800 μ g/ml), MPA (0.10; 0.25,2.00,7.50 μ g/ml) and MPAG (20,40; 90,150 μ g/ml) urine quality-control product, by " the urine 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 urine 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 12%, deviation is less than 9%.
Embodiment 2: renal transplant recipients urine sample 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 urine sample pre-service
The accurate 100 μ L urine samples of drawing are put in the 1.5mL centrifuge tube, add methanol solution 500 μ L dilution, vortex vibration 15s, and centrifugal 5min (20,627 * g, 4 ℃) gets supernatant 20 μ L sample introductions, and external standard method is with peak area quantification.
Specificity
Get 8 blank urines 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 6.4,15.6,4.9 and 10.9min, and whole stratographic analysis process time is 17.5min.
Linear test
Get blank urine, add an amount of MMF, MPA and MPAG standard reserving solution are mixed with and contain MMF (0.075,0.125,0.250 respectively; 0.500,0.750,1.000 μ g/ml), MPA (0.1,0.5; 1.0,2.5,5.0,10.0 μ g/ml) and MPAG (20,50; 80,100,200,400 μ g/ml) urine standard items, by " the urine sample pre-service " the method operation.External standard method is made weighting (1/C with the peak area (A) and the concentration of component to be measured (C) of component to be measured
2) linear regression, three's typical curve regression equation is respectively: MMF C=7.6546 * 10
-6A-0.0024, r=0.9993; MPA8.4023 * 10
-6A+0.0019, r=0.9994; MPAG C=0.0013A+5.4853, r=0.9995.
Accuracy and precision
Get blank urine, add an amount of MMF, MPA and MPAG standard reserving solution are mixed with and contain MMF (0.075 respectively; 0.150,0.400,0.800 μ g/ml), MPA (0.10; 0.25,2.00,7.50 μ g/ml) and MPAG (20,40; 90,150 μ g/ml) urine quality-control product, by " the urine 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 urine 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 13%, deviation is less than 10%.
Table 1
Table 2
Claims (2)
1. method of measuring mycophenolate in the human urine, 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, described metabolin is mycophenolic acid glucoside acidifying thing MPAG; This method may further comprise the steps:
1) sample pretreatment
Get testing sample, add methyl alcohol, deionized water or both mixed liquors, carry out the urine sample dilution, get the supernatant sample introduction after centrifugal;
2) sample separation and detection MPAG
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 UV-detector to detect the peak area of MPAG, be converted into concentration with the typical curve equation;
Wherein chromatographic condition is, the LC-10A of HPLC system high performance liquid chromatogram appearance; Chromatographic column: C8 analytical column; Moving phase: adopt isocratic elution, methyl alcohol-0.1%TFA 50: 50-60: 40, and 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 and detection mycophenolate and mycophenolic acid
Behind UV-detector; Adopt derivatization reagent behind an additional high-pressure pump and the threeway joint pin; And connect UV-detector and fluorescence detector with series system, and carry out the sample post column derivatization, make the sample after the separation that fluorescence signal arranged; Adopt fluorescence detector to detect the peak area of mycophenolate and mycophenolic acid respectively, be converted into concentration with the typical curve equation.
2. the method for measuring mycophenolate in the human urine, 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 urine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1619304A (en) * | 2004-09-20 | 2005-05-25 | 复旦大学 | Method of determining mycophenolic acid in human blood plasma and its metabolite |
| CN1971270A (en) * | 2005-11-24 | 2007-05-30 | 复旦大学附属华山医院 | Method for detecting blood concentration of multiple antiepileptic drugs simultaneously |
| CN101105479A (en) * | 2007-08-10 | 2008-01-16 | 复旦大学附属华山医院 | Method for determining human plasma phenytoin and its precursor drug and metabolite |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1619304A (en) * | 2004-09-20 | 2005-05-25 | 复旦大学 | Method of determining mycophenolic acid in human blood plasma and its metabolite |
| CN1971270A (en) * | 2005-11-24 | 2007-05-30 | 复旦大学附属华山医院 | Method for detecting blood concentration of multiple antiepileptic drugs simultaneously |
| CN101105479A (en) * | 2007-08-10 | 2008-01-16 | 复旦大学附属华山医院 | Method for determining human plasma phenytoin and its precursor drug and metabolite |
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