CN100414293C - Method for determining blood drug level of mizoribine - Google Patents
Method for determining blood drug level of mizoribine Download PDFInfo
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- CN100414293C CN100414293C CNB2006100233859A CN200610023385A CN100414293C CN 100414293 C CN100414293 C CN 100414293C CN B2006100233859 A CNB2006100233859 A CN B2006100233859A CN 200610023385 A CN200610023385 A CN 200610023385A CN 100414293 C CN100414293 C CN 100414293C
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- acetonitrile
- methyl alcohol
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- mizoribine
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Abstract
The present invention relates to a method for analyzing and measuring medicines in a body, particularly to a method for measuring mizoribine concentration in human plasma. The present invention belongs to the medicine test field. After albumen precipitation pretreatment is carried out on a sample to be measured with the method of the present invention, isocratic elution is carried out under the condition of an acidic mobile phase, and an uv detector is used for detection after the separation of a chromatographic column. The method has the advantages of few sample taking quantities, simple pretreatment method, quickness, sensitivity without expensive equipment and reagents, wide application scope and low cost and is suitable for monitoring the conventional blood concentration of a clinic.
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 blood drug level of mizoribine.
Background technology
(mizoribine MZR) is a kind of purine nucleosides synthetic inhibitor to mizoribine, can suppress the lymphocyte of growth fast specifically, thereby produces immunosuppressive action.Rejection when being used to suppress kidney transplant clinically in recent years also is used for the treatment of autoimmune diseases such as lupus nephritis, rheumatoid arthritis and nephrotic syndrome simultaneously.Pharmacokinetic differences is big between the individuality of this medicine, must carry out therapeutic drug monitoring clinically to adjust the individual administration scheme.
About the assay method of MZR in the biological specimen, relevant report is only arranged abroad at present.There is low (the minimum 0.25mgL that quantitatively is limited to of sensitivity in existing technical method
-1), many disadvantages such as complex operation is time-consuming, efficient is low, analysis cost height, be not suitable for the conventional therapy monitor drug concentration.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, the method for blood drug level of mizoribine in a kind of easy, quick, sensitive mensuration body is provided.
This method need not expensive equipment and reagent, has easy and simple to handle, quick, few, the low cost and other advantages of blood plasma consumption, is fit to conventional therapeutic drug monitoring.
The technical scheme of this method is: after the testing sample pre-service, separate in chromatographic column through acid moving phase, detect with UV-detector.
Comprise the steps:
1) sample pretreatment
Get testing sample, add quantitative organic solvent protein precipitant and carry out albumen precipitation,
Described organic solvent protein precipitant is selected from the potpourri of methyl alcohol, acetonitrile or methyl alcohol and acetonitrile;
2) sample separation and analysis
Adopt universal liquid-phase chromatographic column, its filler is Phenomenex Luna NH
2, the high performance liquid chromatogram system adopts universal UV-detector and high-pressure pump, and the mixed liquor that adopts methyl alcohol, acetonitrile and acidic aqueous solution (pH=2.1～2.5) is as moving phase, isocratic elution;
This method moving phase particular methanol-acetonitrile-0.1% trifluoroacetic acid (85 ± 2: 5 ± 1: 10 ± 1) mixed liquor;
Adopt UV-detector 280 ± 1nm to detect the peak area of MZR, be converted into concentration with the typical curve equation.
This method has the following advantages:
1. preprocess method is easy: a step organic solvent precipitation of protein is applicable to conventional sense.
2. specificity is strong: adopting filler is Phenomenex Luna NH
2Chromatographic column as stationary phase, the mixed liquor of methyl alcohol, acetonitrile and acidic aqueous solution is as moving phase, isocratic elution was finished mensuration in 12 minutes, endogenous material and drug combination commonly used do not disturb the mensuration of said medicine.
3. highly sensitive: the minimum 0.1mgL that quantitatively is limited to
-1, be better than the 0.25mgL of bibliographical information
-1
The inventive method quick and precisely, highly sensitive, easy and simple to handle, cost is low, be suitable for routine clinical therapeutic drug monitoring.The range of linearity of MZR is 0.1～10mgL
-1The averaging method recovery is respectively 98.7%, in a few days with day to day precision RSD all less than 9%.
Description of drawings
Fig. 1 is the typical color spectrogram,
Wherein, A: the renal transplant recipients blood plasma of not taking mizoribine; B: blank plasma+standard items mizoribine (concentration: 0.1mgL
-1); 1: mizoribine.
Fig. 2 is the renal transplant recipients chromatogram (concentration: 0.26mgL of taking mizoribine
-1),
Wherein, 1: mizoribine.
Embodiment
Chromatographic condition
Chromatographic column: Phenomenex Luna NH
2(4.6mm * 250mm, 5 μ m, U.S. Phenomenex company); Moving phase: acetonitrile-methyl alcohol-0.1% trifluoroacetic acid (85: 5: 10); Flow velocity: 1.5mLmin
-1Column temperature: 30 ℃; Detect wavelength, 280nm.
The plasma sample pre-service
Get plasma sample 100 μ L, add acetonitrile liquid 100 μ L, vortex mixed 20s, the centrifugal 10min of 12000 * g under 4 ℃ of conditions gets supernatant 20 μ L sample introductions.External standard method is with peak area quantification.
Specificity
Get 10 renal transplant recipients blood samples of not taking MZR of separate sources, measure according to above-mentioned sample pretreatment and assay method, the result shows, does not find that endogenous material has interference to the said determination component.Get drug combination commonly used and OTC (over-the-counter) reference substance solution sample introduction, the result shows, cyclosporine, prednisone, Ta Mokesi, Xi Luomosi, Ganciclovir, times Ta Luoke, paracetamol, nifedipine, brufen, Acyclovir etc. all not disturbed specimen measure.Its typical retention time is 7.6min.Whole stratographic analysis process time is 12min.
Linear test
It is an amount of that precision takes by weighing standard substance, becomes serial working fluid with dissolved in distilled water, adds an amount of blank human plasma again, makes to contain MZR 0.1,0.5,2.5,5,7.5,10mgL
-1The standard blood sample.Get blood plasma 100 μ L, by " the plasma sample pre-service " the method operation.External standard method is done weighting (1/x) linear regression with MZR peak area (x) and MZR concentration (y), gets equation of linear regression to be: y=9171.3x-21.6, (r=0.9996).The MZR plasma concentration is at 0.1～10mgL
-1Scope internal linear relation is good.Minimum quantitative concentrations is 0.1mgL
-1
Accuracy and precision
It is an amount of that precision takes by weighing standard substance, uses dissolved in distilled water, and blank plasma dilutes constant volume, makes to contain MZR 0.2,4,8mgL
-1Basic, normal, high Quality Control blood sample.Get blood plasma 100 μ L,, investigate in a few days and day to day precision and accuracy by " plasma sample pre-service " method operation.Calculate its measured concentration according to equation of linear regression, calculate the actual measurement mean value and the relative standard deviation (RSD) of every kind of concentration, (C
Actual measurement/ C
Theoretical) * 100% is the recovery, and experimental result shows: endogenous material is not disturbed the mensuration of determinand.And above-mentioned component to be measured and internal standard compound mass peak shape symmetry, separate good.Table 1 be mizoribine in a few days, day to day precision and the method recovery.
Simultaneously, this method has obtained checking in the mensuration of actual patient blood concentration.
Table 1
Embodiment 2
Chromatographic condition
Chromatographic column: Phenomenex Luna NH
2(4.6mm * 250mm, 5 μ m, U.S. Phenomenex company); Moving phase: acetonitrile-methyl alcohol-0.1% trifluoroacetic acid (85: 5: 10); Flow velocity: 1.5mLmin
-1Column temperature: 30 ℃; Detect wavelength, 280nm.
The plasma sample pre-service
Get plasma sample 100 μ L, and the mixed liquor of adding acetonitrile and methyl alcohol (1: 1, v/v) 100 μ L, vortex mixed 20s, the centrifugal 10min of 12000 * g under 4 ℃ of conditions gets supernatant 20 μ L sample introductions.External standard method is with peak area quantification.
Specificity
Get 10 renal transplant recipients blood samples of not taking MZR, measure according to above-mentioned sample pretreatment and assay method.Other gets drug combination commonly used and OTC (over-the-counter) reference substance solution sample introduction.The result show endogenous material, cyclosporine, prednisone, Ta Mokesi, Xi Luomosi, Ganciclovir, times Ta Luoke, paracetamol, nifedipine, brufen, Acyclovir etc. all not disturbed specimen measure.Its typical retention time is 7.6min.Whole stratographic analysis process time is 12min.
Linear test
It is an amount of that precision takes by weighing standard substance, becomes serial working fluid with dissolved in distilled water, adds an amount of blank human plasma again, makes to contain MZR 0.1,0.5,2.5,5,7.5,10mgL
-1The standard blood sample.Get blood plasma 100 μ L, by " the plasma sample pre-service " the method operation.External standard method is done weighting (1/x) linear regression with MZR peak area (x) and MZR concentration (y), gets equation of linear regression to be: y=9082.4x-19.1, (r=0.9996).The MZR plasma concentration is at 0.1～10mgL
-1Scope internal linear relation is good.Minimum quantitative concentrations is 0.1mgL
-1Minimal detectable concentration is 0.3mgL
-1
Accuracy and precision
It is an amount of that precision takes by weighing standard substance, uses dissolved in distilled water, and blank plasma dilutes constant volume, makes to contain MZR 0.2,4,8mgL
-1Basic, normal, high Quality Control blood sample.Get blood plasma 100 μ L, by " the plasma sample pre-service " method operation, investigate in a few days and day to day precision and accuracy (n=6).Calculate its measured concentration according to equation of linear regression, calculate the actual measurement mean value and the relative standard deviation (RSD) of every kind of concentration, (C
Actual measurement/ C
Theoretical) * 100% is the recovery.Experimental result shows: method in a few days with day to day precision all less than 10%, relative standard deviation is less than 8%.
Embodiment 3
Chromatographic condition
Chromatographic column: Phenomenex Luna NH
2(4.6mm * 250mm, 5 μ m, U.S. Phenomenex company); Moving phase: acetonitrile-methyl alcohol-0.1% trifluoroacetic acid (85: 5: 10); Flow velocity: 1.5mLmin
-1Column temperature: 30 ℃; Detect wavelength, 280nm.
The plasma sample pre-service
Get plasma sample 100 μ L, add methanol solution 200 μ L, vortex mixed 20s, the centrifugal 10min of 12000 * g under 4 ℃ of conditions gets supernatant 30 μ L sample introductions.External standard method is with peak area quantification.
Specificity
Get 10 renal transplant recipients blood samples of not taking MZR, measure according to above-mentioned sample pretreatment and assay method.Other gets drug combination commonly used and OTC (over-the-counter) reference substance solution sample introduction.The result show endogenous material, cyclosporine, prednisone, Ta Mokesi, Xi Luomosi, Ganciclovir, times Ta Luoke, paracetamol, nifedipine, brufen, Acyclovir etc. all not disturbed specimen measure.Its typical retention time is 7.6min.Whole stratographic analysis process time is 12min.
Linear test
It is an amount of that precision takes by weighing standard substance, becomes serial working fluid with dissolved in distilled water, adds an amount of blank human plasma again, makes to contain MZR 0.1,0.5,2.5,5,7.5,10mgL
-1The standard blood sample.Get blood plasma 100 μ L, by " the plasma sample pre-service " the method operation.External standard method is done weighting (1/x) linear regression with MZR peak area (x) and MZR concentration (y), gets equation of linear regression to be: y=9174.8x-27.6, (r=0.9994).The MZR plasma concentration is at 0.1～10mgL
-1Scope internal linear relation is good.Minimum quantitative concentrations is 0.1mgL
-1Minimal detectable concentration is 0.3mgL
-1
Accuracy and precision
It is an amount of that precision takes by weighing standard substance, uses dissolved in distilled water, and blank plasma dilutes constant volume, makes to contain MZR 0.2,4,8mgL
-1Basic, normal, high Quality Control blood sample.Get blood plasma 100 μ L, by " the plasma sample pre-service " method operation, investigate in a few days and day to day precision and accuracy (n=6).Calculate its measured concentration according to equation of linear regression, calculate the actual measurement mean value and the relative standard deviation (RSD) of every kind of concentration, (C
Actual measurement/ C
Theoretical) * 100% is the recovery.Experimental result shows: method in a few days with day to day precision all less than 10%, relative standard deviation is less than 8%.
Claims (1)
1. method of measuring blood drug level of mizoribine, it is characterized in that the testing sample pre-service after, separate on chromatographic column through acid moving phase, detect with UV-detector, comprise the steps:
1) sample pretreatment
Get testing sample, add a kind of in quantitative methyl alcohol, acetonitrile or methyl alcohol and the acetonitrile potpourri, carry out albumen precipitation;
2) sample separation
Adopt universal liquid-phase chromatographic column, its filler is Phenomenex Luna NH
2The high performance liquid chromatogram system adopts universal UV-detector and high-pressure pump, described acid moving phase adopts the mixed liquor of methyl alcohol-acetonitrile-0.1% trifluoroacetic acid aqueous solution, described methyl alcohol: acetonitrile: 0.1% trifluoroacetic acid aqueous solution is 85 ± 2: 5 ± 1: 10 ± 1, flow velocity is 1.5mL/min, isocratic elution;
3) UV-detector detects: the detection wavelength is 280 ± 1nm, surveys peak area, with typical curve equation conversion concentration.
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| CNB2006100233859A CN100414293C (en) | 2006-01-16 | 2006-01-16 | Method for determining blood drug level of mizoribine |
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| CN100414293C true CN100414293C (en) | 2008-08-27 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100580447C (en) * | 2007-07-31 | 2010-01-13 | 复旦大学附属华山医院 | Method for determining human plasma antiviral drug concentration |
| CN104483493B (en) * | 2014-12-30 | 2016-09-28 | 青岛市市立医院 | A kind of mizoribine that screens is in the method for immune t-cell Chinese medicine target site |
| CN114062557B (en) * | 2021-11-19 | 2023-07-25 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Detection method for degradation impurities in mizoribine bulk drug |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6140363A (en) * | 1998-05-16 | 2000-10-31 | Mogam Biotechnology Research Institute | Use of rosmarinic acid and derivatives thereof as an immunosuppressant or an inhibitor of SH2-mediated processes |
| CN1178652C (en) * | 2000-01-26 | 2004-12-08 | 旭化成制药株式会社 | Mizoribine tablet with improved color |
-
2006
- 2006-01-16 CN CNB2006100233859A patent/CN100414293C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6140363A (en) * | 1998-05-16 | 2000-10-31 | Mogam Biotechnology Research Institute | Use of rosmarinic acid and derivatives thereof as an immunosuppressant or an inhibitor of SH2-mediated processes |
| CN1178652C (en) * | 2000-01-26 | 2004-12-08 | 旭化成制药株式会社 | Mizoribine tablet with improved color |
Non-Patent Citations (1)
| Title |
|---|
| Implication if the peak mizoribine for control of serum level ofthe serum anti-dsDNA antibody titer in patients with iupusnephritis. Tanaka H etal.CLINICAL NEPHROLOGY,Vol.63 No.6. 2005 * |
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