CN102384949A - Method for simultaneously measuring content of three antitubercular agents in blood and tissues - Google Patents
Method for simultaneously measuring content of three antitubercular agents in blood and tissues Download PDFInfo
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- CN102384949A CN102384949A CN2011102282174A CN201110228217A CN102384949A CN 102384949 A CN102384949 A CN 102384949A CN 2011102282174 A CN2011102282174 A CN 2011102282174A CN 201110228217 A CN201110228217 A CN 201110228217A CN 102384949 A CN102384949 A CN 102384949A
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Abstract
The invention provides a method for simultaneously measuring content of three antitubercular agents (retozide, rifampicin and pyrazinamide) in blood and tissues. The method has the characteristics of simplicity and convenience in operation, high precision, high detection speed, stability and reliability. A high-efficient liquid-phase chromatographic instrument (LC2010C), a column temperature control box AT-130 and an Lc-solution spectrum work station of the Japan Shimadzu Company are adopted. The chromatographic column is an analysis column and adopts a C8 column, the protection column adopts a C18 column, and the fluid phase adopts sodium 1-heptanesulfonate solution to prepare.
Description
Affiliated field:
The present invention be belong to a kind of measure simultaneously in the method for three kinds of antituberculotics (isoniazid, rifampin, pyrazinamide) content, specifically a kind ofly relate to the method that high pressure lipuid chromatography (HPLC) is measured three kinds of antituberculotic content in blood and the tissue simultaneously.
Background technology:
Isoniazid (Isoniazid); Rifampin (Rifampicin); Pyrazinamide (Pyrazinamide) is still a current widely used line antituberculotic, and the isoniazid has neurotoxicity, and rifampin has hepatotoxicity wind agitation, and therefore antituberculotic is carried out concentration monitor has crucial clinical meaning.The physicochemical property of these three kinds of medicines is completely contradicted, and isoniazid and pyrazinamide are strong polar material, and water-soluble big, and rifampin is the low pole material, and is fat-soluble big, therefore will be under a chromatographic condition three kinds of medicines be separated fully to have certain difficulty.Existing monitoring method mainly adopts phosphate buffer as moving phase; Adopt different chromatographic column (C8 post and C18 post) under different chromatographic conditions, to detect this three kinds of content of medicines respectively, or part medicine is wherein carried out derivatization sample introduction monitoring more in advance.This class methods technology is very complicated comparatively, and laboratory condition is had relatively high expectations, and needs repeatedly duplicate detection, and the time that goes out examining report is also longer, and is also bigger to the requirement of sample size, increased the difficulty of Clinical detection.
Summary of the invention:
The object of the invention just provides a kind of easy and simple to handle, and degree of accuracy is high, and detection speed is fast, and is reliable and stable, can measure the method for three kinds of antituberculotic content simultaneously.
The detection method of invention comprises the steps:
(1) sample preparation
Blood sample adopts (4000 rev/mins) to get blood plasma after centrifugal 15 minutes, and (4000 rev/mins) were centrifugal after tissue samples adding physiological saline adopted homogenizer to smash to pieces, added extract and extracted.
(2) chromatographic condition
Moving phase is 0.02mol/L, and heptanesulfonic acid sodium solution (phosphoric acid,diluted is transferred pH to 3.0)-acetonitrile-methyl alcohol filtered the back ultrasonic 15 minutes before moving phase is used, the degassing.
(3) sample analysis
Adopt day island proper Tianjin company's high performance liquid chromatograph (LC2010C), AT-130 post temperature control box, Lc-solution type chromatographic work station.Chromatographic column is an analytical column: shim-pack HRC-C8 (250mm * 4.6mm, 5 μ m), and guard column: Eclipse XDB-C18 (125mm * 4.6mm), column temperature 25C °.
(4) set up typical curve
Blank plasma added to be diluted to behind the standard solution containing the standard blood plasma that INH, RPF, PZA are respectively 0.1,0.2,0.4,1,2,4,8,16 μ g/ml, is that X axle, three kinds of drug concentrations are that the Y axle is done linear analysis with the peak area of three kinds of medicines.
Get the regression equation of three kinds of medicines:
Y
The isoniazid=2370.46X+1282.66
Y
Pyrazinamide=86447.8X+5195.94
Y
Rifampin=3007.2X+3714.58
Chromatogram medicine peak area behind the sample sample introduction is brought in the above formula, can calculates drug concentration.
Advantage of the present invention is:
1. broad applicability: this chromatography not only can be used for blood, can also be used for the detection of three kinds of antituberculotic content in the biopreparate samples such as other body fluid of human body and tissue, newborn saliva, urine, cerebrospinal fluid, seminal fluid etc.As long as can liquid condition can in the tissue separated all can detect, can be the detection of clinical blood concentration, the research of pharmacokinetics, the adjustment of therapeutic regimen etc. provides the reliable detection means.Therefore broad applicability is that this chromatographic detection method has far-reaching prospect.
2. the simplicity of sample process: this chromatographic process need not to carry out derivatization in advance to the processing of sample and simple.Get serum after Blood Preparations is directly centrifugal and extract with extract, tissue samples is centrifugal after adopting even matter device to smash to pieces, gets supernatant and extracts with extract.Method is simple, and the processing time is short, the convenient row that is prone to.
3. testing process is quick: this chromatographic process adopts the method for gradient elution that sample is detected; Can detect three kinds of drug concentrations in the sample simultaneously by single injected sampling; Weak point consuming time, retention time were less than 30 minutes, and promptly single injected sampling just can draw testing result in 30 minutes.Because single injected sampling can detect three kinds of medicament contgs, compare repeatedly sample introduction and detect of the consumption also greatly reduction of three kinds of medicines respectively simultaneously detecting sample.
4. testing result accurate and stable: this chromatographic process precision stable (pyrazinamide: 8.4%, the isoniazid: 10.1%, rifampin: 9.5%.), the recovery high (pyrazinamide: 83.7%, the isoniazid: 86.2%, rifampin: 88.1%.)
1.1 test sample is a blood
1.2 chromatographic condition:
Chromatographic column is analytical column shim-pack HRC-C8 (250mm * 4.6mm, 5 μ m), and guard column Eclipse XDB-C18 (125mm * 4.6mm), column temperature 25C °; Moving phase is 0.02mol/L, heptanesulfonic acid sodium solution (phosphoric acid,diluted is transferred pH to 3.0)-acetonitrile-methyl alcohol (78: 5: 17) 6min, (20: 5: 75) 16min; (78: 5: 17) 27min. flow velocity is 0.8ml/min; Test wavelength pyrazinamide: 268nm, isoniazid: 264nm, rifampin: the 254nm. retention time is pyrazinamide: 5.62min respectively; Isoniazid: 10.01min, rifampin: 24.89min.
1.3 stratographic analysis result:
Chromatographic resolution is all right, and the endogenous magazine does not disturb mutually in three kinds of medicines and the blood.Fig. 1 is the spectrogram behind the blank plasma sample introduction; Fig. 2 adds the chromatogram of sample introduction behind three kinds of medicines for blank plasma; Fig. 3 is the chromatogram behind the blood sample sample introduction, after Fig. 5-7 adds three kinds of medicines of variable concentrations for blank plasma, and the linear relationship of its concentration and peak area.
1.4 calculating medicament contg
Do to get after the linear analysis according to Fig. 5-7, draw the regression equation of three medicines according to medicine peak area and concentration relationship
Y
The isoniazid=2370.4X+1282.66
Y
Pyrazinamide=86447.8X+5195.94
Y
Rifampin=3007.2X+3714.58
With the medicine peak area of the chromatogram behind Fig. 3 blood sample sample introduction, isoniazid: 6426.56m
2, rifampin: 16976.33m
2, pyrazinamide: 2060924.62m
2Be brought in the middle of the above-mentioned equation, can calculate three kinds of content of medicines, the isoniazid: 2.17 μ g/ml, rifampin: 4.41 μ g/ml, pyrazinamide: 23.78 μ g/ml.
2.1 test sample is a bone
2.2 chromatographic condition:
Chromatographic column is analytical column shim-pack HRC-C8 (250mm * 4.6mm, 5 μ m), and guard column Ecl ipse XDB-C18 (125mm * 4.6mm), column temperature 25C °; Moving phase is 0.02mol/L, heptanesulfonic acid sodium solution (phosphoric acid,diluted is transferred pH to 3.0)-acetonitrile-methyl alcohol (78: 5: 17) 6min, (20: 5: 75) 16min; (78: 5: 17) 27min. flow velocity is 0.8ml/min; Test wavelength pyrazinamide: 268nm, isoniazid: 264nm, rifampin: the 254nm. retention time is pyrazinamide: 5.62min respectively; Isoniazid: 10.01min, rifampin: 24.89min.
2.3 stratographic analysis result:
Chromatographic resolution is all right, and the endogenous magazine does not disturb mutually in three kinds of medicines and the blood.Fig. 1 is the spectrogram behind the blank plasma sample introduction; Fig. 2 adds the chromatogram of sample introduction behind three kinds of medicines for blank plasma; Fig. 4 is the chromatogram behind the sample bone sample introduction, after Fig. 5-7 adds three kinds of medicines of variable concentrations for blank plasma, and the linear relationship of its concentration and peak area.
1.4 calculating medicament contg
Do to get after the linear analysis according to Fig. 5-7, draw the regression equation of three medicines according to medicine peak area and concentration relationship
Y
The isoniazid=2370.4X+1282.66
Y
Pyrazinamide=86447.8X+5195.94
Y
Rifampin=3007.2X+3714.58
With the medicine peak area of the chromatogram behind Fig. 3 blood sample sample introduction, isoniazid: 3321.26m
2, rifampin: 12473.36m
2, pyrazinamide: 559326.34m
2Be brought in the middle of the above-mentioned equation, can calculate three kinds of content of medicines, the isoniazid: 0.86 μ g/ml, rifampin: 2.42 μ g/ml, pyrazinamide: 6.41 μ g/ml
Description of drawings:
Fig. 1 is the spectrogram behind the blank plasma sample introduction
Fig. 2 is the chromatogram of sample introduction behind three kinds of medicines of blank plasma interpolation
Fig. 3 is the chromatogram behind the blood sample sample introduction
Fig. 4 is the chromatogram behind the sample bone sample introduction
Fig. 5 is isoniazid drug concentration and peak area linear relationship chart
Fig. 6 is pyrazinamide drug concentration and peak area linear relationship chart
Fig. 7 is rifampicin medicine concentration and peak area linear relationship chart.
Claims (4)
1. a high pressure lipuid chromatography (HPLC) is measured the method for three kinds of approach (isoniazid, rifampin, pyrazinamide) medicament contg in blood and the tissue simultaneously, it is characterized in that comprising the steps:
(1) sample preparation
Blood sample adopts (4000 rev/mins) to get blood plasma after centrifugal 15 minutes, and (4000 rev/mins) were centrifugal after tissue samples adding physiological saline adopted homogenizer to smash to pieces, added extract and extracted.
(2) chromatographic condition
Moving phase is 0.02mol/L, and heptanesulfonic acid sodium solution (phosphoric acid,diluted is transferred pH to 3.0)-acetonitrile-methyl alcohol filtered the back ultrasonic 15 minutes before moving phase is used, the degassing.
(3) sample analysis
Adopt day island proper Tianjin company's high performance liquid chromatograph (LC2010C), AT-130 post temperature control box, Lc-solution type chromatographic work station.Chromatographic column is an analytical column: shim-pack HRC-C8 (250mm * 4.6mm, 5 μ m), and guard column: Eclipse XDB-C18 (125mm * 4.6mm), 25 ℃ of column temperatures.
(4) set up typical curve
Blank plasma added to be diluted to behind the standard solution containing the standard blood plasma that INH, RPF, PZA are respectively 0.1,0.2,0.4,1,2,4,8,16 μ g/ml, is that X axle, three kinds of drug concentrations are that the Y axle is done linear analysis with the peak area of three kinds of medicines.
Get the regression equation of three kinds of medicines:
Y
The isoniazid=2370.4X+1282.66
Y
Pyrazinamide=86447.8X+5195.94
Y
Rifampin=3007.2X+3714.58
Chromatogram medicine peak area behind the sample sample introduction is brought in the above formula, can calculates drug concentration.
2. measure the method for three kinds of antituberculotic content in blood and the tissue according to the said high pressure lipuid chromatography (HPLC) of claim 1 simultaneously, it is characterized in that extract adopts (methylene chloride: isopropyl alcohol=1: 1) dispose, extract.
3. measure the method for three kinds of antituberculotic content in blood and the tissue simultaneously according to the said high pressure lipuid chromatography (HPLC) of claim 1; The ratio that it is characterized in that moving phase is (78: 5: 17) 6min; (20: 5: 75) 16min; (78: 5: 17) 27min, the method for employing gradient elution, flow velocity is 0.8ml/min.
4. measure the method for three kinds of antituberculotic content in blood and the tissue according to the said high pressure lipuid chromatography (HPLC) of claim 1 simultaneously, it is characterized in that testing wavelength PZA:268nm, INH:264nm, RFP:254nm.Flow velocity is 1.5 ml/min, and sampling volume is that 20 microlitres carry out stratographic analysis.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107238672A (en) * | 2017-08-10 | 2017-10-10 | 沈阳红旗制药有限公司 | The content assaying method of impurity in a kind of isoniazid or its pharmaceutical composition |
| CN107941980A (en) * | 2017-11-26 | 2018-04-20 | 浙江省水产技术推广总站 | The remaining ultra performance liquid chromatography tandem mass spectrum rapid assay methods of rifampin in aquatic products |
| CN108226323A (en) * | 2017-12-13 | 2018-06-29 | 重庆华邦胜凯制药有限公司 | A kind of method for measuring isoniazid starting material 4- cyanopyridines and its impurity content |
| CN109580806A (en) * | 2018-11-09 | 2019-04-05 | 佛山科学技术学院 | One kind is for the remaining measuring method of rifampicin medicine in aquatic products |
| CN109682915A (en) * | 2019-01-16 | 2019-04-26 | 徐州立兴佳正医药科技有限公司 | A kind of method that LC-MS measures pyrazinamide concentration in blood plasma |
-
2011
- 2011-08-02 CN CN2011102282174A patent/CN102384949A/en active Pending
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| 刘鹏: "高效液相色谱法测定脊柱结核中抗结核药物浓度的研究", 《中国优秀硕士学位论文全文数据库》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107238672A (en) * | 2017-08-10 | 2017-10-10 | 沈阳红旗制药有限公司 | The content assaying method of impurity in a kind of isoniazid or its pharmaceutical composition |
| CN107238672B (en) * | 2017-08-10 | 2019-06-07 | 沈阳红旗制药有限公司 | The content assaying method of impurity in a kind of isoniazid or its pharmaceutical composition |
| CN107941980A (en) * | 2017-11-26 | 2018-04-20 | 浙江省水产技术推广总站 | The remaining ultra performance liquid chromatography tandem mass spectrum rapid assay methods of rifampin in aquatic products |
| CN108226323A (en) * | 2017-12-13 | 2018-06-29 | 重庆华邦胜凯制药有限公司 | A kind of method for measuring isoniazid starting material 4- cyanopyridines and its impurity content |
| CN109580806A (en) * | 2018-11-09 | 2019-04-05 | 佛山科学技术学院 | One kind is for the remaining measuring method of rifampicin medicine in aquatic products |
| CN109682915A (en) * | 2019-01-16 | 2019-04-26 | 徐州立兴佳正医药科技有限公司 | A kind of method that LC-MS measures pyrazinamide concentration in blood plasma |
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Application publication date: 20120321 |