CN101226172A - Method for rapidly measuring mizoribine drug concentration - Google Patents
Method for rapidly measuring mizoribine drug concentration Download PDFInfo
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- CN101226172A CN101226172A CNA2007100263365A CN200710026336A CN101226172A CN 101226172 A CN101226172 A CN 101226172A CN A2007100263365 A CNA2007100263365 A CN A2007100263365A CN 200710026336 A CN200710026336 A CN 200710026336A CN 101226172 A CN101226172 A CN 101226172A
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- mizoribine
- blood drug
- drug level
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Abstract
The invention relates to an analysis measurement method of internal drug, in particular to a method for quickly measuring mizonbine density of human blood plasma, which comprises adding sample into an interior label, after protein deposition pretreatment, isocratically eluting under water-type flow phase condition, separating via a chromatographic column, and detecting via an ultrasonic detector. The invention has low sample consumption, simple, quick and sensitive pretreatment, which not demands expensive device and organic solvent, with wide application, low environment pollution, low cost and the application for detecting clinical general blood drug density.
Description
Technical field
The invention belongs to field of medical examination, relate to the analysis determining method of drug disposition, be specifically related to the method for mizoribine concentration in a kind of fast measuring human plasma.
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.
At present about the assay method of MZR in the biological specimen, both at home and abroad relevant report seldom, existing method does not all adopt inner mark method ration, accuracy of measurement, precision are lower, and have low (the minimum 0.1mgL that quantitatively is limited to of sensitivity
-1), many disadvantages such as complex operation is bothersome, 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, need not organic solvent, has advantages such as easy and simple to handle, quick, that the blood plasma consumption is few, cost is low, environmental pollution is little, is fit to conventional therapeutic drug monitoring.
The technical scheme of this method is: mark in testing sample adds, after acidic precipitation agent pre-service, separate in chromatographic column through water-based moving phase, and detect with UV-detector.
Comprise the steps:
1) sample pretreatment
Get testing sample, albumen precipitation is carried out with the acidic precipitation agent in the mark back in adding,
Be designated as cytarabine in described, the acidic protein precipitation agent is 0.1-0.5molL
-1Perchloric acid solution;
2) sample separation and analysis
Adopt universal liquid-phase chromatographic column, its filler is Hypersil BDS C18 5um, and the high performance liquid chromatogram system adopts universal UV-detector and high-pressure pump, and moving phase adopts the 10mmolL that contains perchloric acid
-1Potassium phosphate buffer (PH6.0-6.5), isocratic elution:
The preferred perchloric acid concentration of this method moving phase is 5-30mmolL
-1
Adopt UV-detector 280 ± 1nm to detect the peak area of cytarabine and MZR, be converted into concentration with the typical curve equation.
This method has the following advantages:
1, sample pretreatment process is marked in adding, and helps improving the accuracy and the precision of assay method.
2, preprocess method is easy: step acidic precipitation agent precipitation of protein is applicable to conventional sense.
3, specificity is strong: adopt filler be the chromatographic column of Hypersil BDS C18 5um as stationary phase, contain the 10mmolL of perchloric acid
-1Potassium phosphate buffer is as moving phase, and isocratic elution was finished mensuration in 11 minutes, and endogenous material and drug combination commonly used do not disturb the mensuration of said medicine.
4, highly sensitive: as minimumly quantitatively to be limited to: 0.02mgL
-1, be better than the 0.1mgL 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 0.02-10mgL
-1The averaging method recovery is 101.84%, in a few days with day to day precision RSD all less than 7%.
Description of drawings
Fig. 1 is the typical color spectrogram,
A wherein: the renal transplant recipients blood plasma of not taking mizoribine; B: blank plasma+standard items mizoribine (concentration: 0.5mgL
-1)+interior mark cytarabine (concentration: 2mgL
-1);
1: mizoribine; 2: cytarabine.
Fig. 2 is the renal transplant recipients blood plasma chromatogram (concentration: 0.31mgL of taking mizoribine
-1) wherein, 1: mizoribine; 2: cytarabine.
Embodiment
Chromatographic condition
Chromatographic column: Hypersil BDS C18 (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. for 4.6 * 250mm, 5 μ m); Moving phase: 10mmolL
-1Potassium dihydrogen phosphate (contains perchloric acid 10mmolL
-1, PH6.0 6.5); Flow velocity: 1.5mLmin
-1Column temperature: room temperature; Detect wavelength: 280nm.
The plasma sample pre-service
Get plasma sample 200 μ L, add 20mgL
-1Cytarabine 20 μ L add distilled water 20 μ L, and vortex mixed 30s adds 0.1molL
-1Perchloric acid, vortex mixed 1min, 10000rmin
-1Centrifugal 5min gets supernatant 20 μ L sample introductions.Internal standard method is quantitative with MZR and cytarabine peak area ratio.
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, tacrolimus, sirolimus, Mycophenolic Acid, Ismipur, Acyclovir, Ganciclovir, Ribavirin, times Ta Luoke, nifedipine, diltiazem, paracetamol, brufen, Diclofenac etc. all not disturbed specimen measure.MZR typical case retention time is 4.0min, and cytarabine typical case retention time is 6.8min, and the stratographic analysis process time is 11min.
Linear test
It is an amount of that precision takes by weighing standard substance, becomes serial working fluid with dissolved in distilled water, adds blank human plasma again, makes to contain MZR 0.02,0.05,0.1,0.2,0.5,1,2,5,10mgL
-1The standard blood sample.Get blood plasma 200 μ L, by the operation of " plasma sample pre-service " method.Internal standard method is a horizontal ordinate with MZR and cytarabine peak area ratio (x), and MZR concentration (y) is the linear recurrence of ordinate, and getting equation of linear regression is y=0.703x-0.021 (r=0.9998).The MZR plasma concentration is at 0.02-10mgL
-1Scope internal linear relation is good.Minimum quantitative concentrations is 0.02mgL
-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 0.075,0.75,7.5mgL
-1Basic, normal, high Quality Control blood sample.Get blood plasma 200 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.Above-mentioned component to be measured and internal standard compound mass peak shape symmetry, separate good.Table 1 be MZR 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
Chromatographic condition
Chromatographic column: Hypersil BDS C18 (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. for 4.6 * 250mm, 5 μ m); Moving phase: 10mmolL
-1Potassium dihydrogen phosphate (contains perchloric acid 20mmolL
-1, PH6.0-6.5); Flow velocity: 1.5mLmin
-1Column temperature: room temperature; Detect wavelength: 280nm.
The plasma sample pre-service
Get plasma sample 200 μ L, add 20mgL
-1Cytarabine 20 μ L add distilled water 20 μ L, and vortex mixed 30s adds 0.25molL
-1Perchloric acid, vortex mixed 1min, 10000rmin
-1Centrifugal 5min gets supernatant 20 μ L sample introductions.Internal standard method is quantitative with MZR and cytarabine peak area ratio.
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, tacrolimus, sirolimus, Mycophenolic Acid, Ismipur, Acyclovir, Ganciclovir, Ribavirin, times Ta Luoke, nifedipine, diltiazem, paracetamol, brufen, Diclofenac etc. all not disturbed specimen measure.MZR typical case retention time is 4.0min, and cytarabine typical case retention time is 6.8min, and the stratographic analysis process time is 11min.
Linear test
It is an amount of that precision takes by weighing standard substance, becomes serial working fluid with dissolved in distilled water, adds blank human plasma again, makes to contain MZR 0.02,0.05,0.1,0.2,0.5,1,2,5,10mgL
-1The standard blood sample.Get blood plasma 200 μ L, by the operation of " plasma sample pre-service " method.Internal standard method is that horizontal ordinate and MZR concentration (y) are the linear recurrence of ordinate with MZR and cytarabine peak area ratio (x), and getting equation of linear regression is y=0.683x-0.019 (r=0.9999).The MZR plasma concentration is at 0.02-10mgL
-1Scope internal linear relation is good.Minimum quantitative concentrations is 0.02mgL
-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 0.075,0.75,7.5mgL
-1Basic, normal, high Quality Control blood sample.Get blood plasma 200 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: the averaging method recovery is 101.84%, in a few days with day to day precision RSD all less than 7%.
Embodiment 3
Chromatographic condition
Chromatographic column: Hypersil BDS C18 (Dalian is according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. for 4.6 * 250mm, 5 μ m); Moving phase: 10mmolL
-1Potassium dihydrogen phosphate (contains perchloric acid 30mmolL
-1, PH6.0-6.5); Flow velocity: 1.5mLmin
-1Column temperature: room temperature; Detect wavelength: 280nm.
The plasma sample pre-service
Get plasma sample 200 μ L, add 20mgL
-1Cytarabine 20 μ L add distilled water 20 μ L, and vortex mixed 30s adds 0.5molL
-1Perchloric acid, vortex mixed 1min, 10000rmin
-1Centrifugal 5min gets supernatant 20 μ L sample introductions.Internal standard method is quantitative with MZR and cytarabine peak area ratio.
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, tacrolimus, sirolimus, Mycophenolic Acid, Ismipur, Acyclovir, Ganciclovir, Ribavirin, times Ta Luoke, nifedipine, diltiazem, paracetamol, brufen, Diclofenac etc. all not disturbed specimen measure.MZR typical case retention time is 4.0min, and cytarabine typical case retention time is 6.8min, and the stratographic analysis process time is 11min.
Linear test
It is an amount of that precision takes by weighing standard substance, becomes serial working fluid with dissolved in distilled water, adds blank human plasma again, makes to contain MZR 0.02,0.05,0.1,0.2,0.5,1,2,5,10mgL
-1The standard blood sample.Get blood plasma 200 μ L, by the operation of " plasma sample pre-service " method.Internal standard method is that horizontal ordinate and MZR concentration (y) are the linear recurrence of ordinate with MZR and cytarabine peak area ratio (x), and getting equation of linear regression is y=0.691x-0.010 (r=0.9998).The MZR plasma concentration is at 0.02-10mgL
-1Scope internal linear relation is good.Minimum quantitative concentrations is 0.02mgL
-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 0.075,0.75,7.5mgL
-1Basic, normal, high Quality Control blood sample.Get blood plasma 200 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: the averaging method recovery is 100.68%, in a few days with day to day precision RSD all less than 7%.
Claims (6)
1. the method for a fast measuring blood drug level of mizoribine, it is characterized in that the testing sample pre-service after, separate on chromatographic column through water-based moving phase, detect with UV-detector, comprise the steps:
1) sample pretreatment
Get testing sample, behind the mark, add quantitative acidic precipitation agent and carry out albumen precipitation in adding;
2) sample separation and analysis
Adopt universal liquid-phase chromatographic column, its filler is Hypersil BDS C185um, and the high performance liquid chromatogram system adopts universal UV-detector and high-pressure pump, and moving phase adopts the potassium phosphate buffer that contains perchloric acid, isocratic elution;
3) UV-detector detects: survey mizoribine, interior mark peak area, with the two peak area ratio substitution typical curve Equation for Calculating concentration.
2. by the method for the described mensuration blood drug level of mizoribine of claim 1, it is characterized in that described step 1) in be designated as cytarabine.
3. by the method for the described mensuration blood drug level of mizoribine of claim 1, the acidic precipitation agent that it is characterized in that described step 1) is 0.1-0.5molL
-1Perchloric acid solution.
4. by the method for the described mensuration blood drug level of mizoribine of claim 1, it is characterized in that described step 2) the moving phase mixed liquor be 10mmolL
-1Potassium phosphate buffer, it contains perchloric acid concentration is 5-30mmolL
-1
5. by the method for the described mensuration blood drug level of mizoribine of claim 1, it is characterized in that described step 2) moving phase mixed liquor PH6.0-6.5.
6. by the method for the described mensuration blood drug level of mizoribine of claim 1, it is characterized in that the UV-detector of described step 3) detects, the detection wavelength is 280nm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100263365A CN101226172A (en) | 2007-01-17 | 2007-01-17 | Method for rapidly measuring mizoribine drug concentration |
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|---|---|---|---|
| CNA2007100263365A CN101226172A (en) | 2007-01-17 | 2007-01-17 | Method for rapidly measuring mizoribine drug concentration |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949885A (en) * | 2010-08-12 | 2011-01-19 | 东南大学 | Capillary electrophoresis-electrochemiluminescence detection method for spectinomycin |
| CN109061022A (en) * | 2018-08-21 | 2018-12-21 | 浙江金大康动物保健品有限公司 | A kind of oriental wormwood detoxification particles Content of Chlorogenic Acid and Gardenoside Simultaneous Determination method |
| CN111879875A (en) * | 2020-08-06 | 2020-11-03 | 武汉伯瑞恒医药科技有限公司 | Method for determining cytarabine and uridine arabinoside in blood plasma |
| CN114062557A (en) * | 2021-11-19 | 2022-02-18 | 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) | Method for detecting degradation impurities in mizoribine raw material medicine |
-
2007
- 2007-01-17 CN CNA2007100263365A patent/CN101226172A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949885A (en) * | 2010-08-12 | 2011-01-19 | 东南大学 | Capillary electrophoresis-electrochemiluminescence detection method for spectinomycin |
| CN109061022A (en) * | 2018-08-21 | 2018-12-21 | 浙江金大康动物保健品有限公司 | A kind of oriental wormwood detoxification particles Content of Chlorogenic Acid and Gardenoside Simultaneous Determination method |
| CN111879875A (en) * | 2020-08-06 | 2020-11-03 | 武汉伯瑞恒医药科技有限公司 | Method for determining cytarabine and uridine arabinoside in blood plasma |
| CN114062557A (en) * | 2021-11-19 | 2022-02-18 | 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) | Method for detecting degradation impurities in mizoribine raw material medicine |
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Open date: 20080723 |