CN104181270A - Temozolomide suppository content measuring method by utilization of reversed-phase ion-pair chromatography method - Google Patents
Temozolomide suppository content measuring method by utilization of reversed-phase ion-pair chromatography method Download PDFInfo
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Abstract
A temozolomide suppository content measuring method by utilization of a reversed-phase ion-pair chromatography method is disclosed. The measuring method is characterized by comprising: (1) a step of preparing a reference substance solution; (2) a step of preparing a solution of a sample to be measured; and (3) a step of content measuring, namely a step of drawing 10-20 [mu]L of the reference substance solution and 10-20 [mu]L of the solution of the sample to be measured separately, injecting into a high performance liquid chromatograph, recording chromatograms, and calculating the content of the temozolomide in the sample to be measured according to the peak area in the chromatograms.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of assay method of timeazoamine ester suppository content.
Background technology
Cervical carcinoma is one of major malignant tumor of serious threat China WomanHealth, and annual new cases 150,000 account for 1/3 of whole world morbidity.Although the trend of dead data shows China recent years, Mortality of cervix cancer declines to some extent, from sample survey data, still can see the existence of cervical carcinoma district occurred frequently, rural area, Midwest particularly, and patient is tending towards rejuvenation.Along with extensively carrying out of cervical carcinoma screening, increasing early cervical carcinoma patient will be by examination out, for the pharmaceutical requirements of cervical carcinoma early treatment, also becomes increasingly conspicuous.
Timeazoamine ester (Temozolomide Hexyl Ester) belongs to the alkylating agent kind anti-cancer drugs in cytotoxic drug, be easy to see through the physiologic barriers such as skin, mucous membrane, make after the suppository of topical administration, local blood concentration is high, antitumous effect is good, is applicable to the diseases such as treatment cervical carcinoma.
The suppository of preparing with timeazoamine ester has vaginal plug or rectal suppository, and the preparation of these suppositorys adopts existing suppository technology of preparing, as: timeazoamine ester is directly scattered in the matrix having melted and is mixed, pack mold into, cooling rear taking-up.Wherein suppository base material comprises: oleaginous base: as cocoa bean ester, fatty acid ester, sheep oil, paraffin etc.; Water-soluble and hydrophilic matrix: as glycerine, gelatin, polyethylene glycols, polyoxyl 40 stearate class etc.; As required, in the formula of suppository, also can add appropriate surface active material: as Tween 80, carbomer, phosphatide etc.; Appropriate antiseptic: as methyl hydroxybenzoate, ethyl hydroxy benzoate, Nipasol etc.
In prior art, the content detection of timeazoamine ester raw material adopts reversed-phased high performace liquid chromatographic, and the dissolution determination of timeazoamine ester bolt adopts ultraviolet spectrophotometry.The assay that these methods are applied to detect the Temozolomide in suppository product still has some deficits.Because major impurity and catabolite also have absorption in the maximum absorption wave strong point of timeazoamine ester, adopt ultraviolet spectrophotometry can not accurately obtain the actual content of medicine; When adopting Suppository By Hplc assay, difficult point is just how tested component is extracted from preparation, dispel unnecessary interfering material.Therefore the invention provides a kind of method that high performance liquid chromatography detects Temozolomide ester content in timeazoamine ester bolt, the method is highly sensitive, and selectivity is strong, and accuracy is good, to the control of product quality, provides a kind of good method.
Summary of the invention:
The object of the present invention is to provide a kind of method with RP-Ion Pair HPLC timeazoamine ester suppository content, specifically comprise the steps:
(1) preparation of reference substance solution;
(2) preparation of need testing solution;
(3) assay: draw reference substance solution, need testing solution, each 10-20ul, injects high performance liquid chromatograph, records chromatogram, according to the peak area of chromatogram, calculates the content of timeazoamine ester in test sample.
Wherein, described in step (1), the preparation method of reference substance solution is as follows:
Take timeazoamine ester reference substance appropriate, with mobile phase, dissolve and be diluted in every 1ml containing tizolemide ester 0.025-0.075mg solution, obtain.
Wherein, described in step (2), the preparation method of need testing solution is as follows:
Get 10, test sample timeazoamine ester bolt, accurately weighed, chopping, mix, precision takes in right amount (being approximately equivalent to timeazoamine ester 50mg), put in 100ml volumetric flask, add acetonitrile to scale, putting 45 ℃ of water-bath joltings dissolves timeazoamine ester, on shaker, vibrate after 2min, putting into refrigerator freezing (18 ℃) filters after 1 hour immediately again, precision measures the subsequent filtrate 1ml of putting to room temperature, put in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that approximately contains 0.05mg in every 1ml, as need testing solution.
Wherein, described chromatographic condition is as follows:
Chromatographic column: the ODS-2HYPERSIL(250mm * 4.6mm of Thermo company, 5 μ m);
Mobile phase is the mixed solution of acetonitrile-sodium acetate and ammonium acetate, volume ratio 35-45: 55-65, and wherein, the mixed solution of the good ammonium acetate of described sodium acetate contains 0.03-0.05molL
-1sodium acetate and 0.005-0.02molL
-1ammonium acetate, also contains 0.01molL in the mixed solution of the good ammonium acetate of sodium acetate
-1perfluorooctane sulfonate, its pH=2.5;
Flow velocity 0.5-1.5mLmin
-1;
Detection wavelength is 300-350nm;
Sample size: 10-20 μ L;
Column temperature: 30-50 ℃.
The preferred technical scheme of the present invention, comprises the steps:
(1) preparation of reference substance solution
It is appropriate that precision takes timeazoamine ester reference substance, dissolves and be diluted in every 1ml to contain tizolemide ester 0.05mg solution with mobile phase, obtain,
(2) preparation of need testing solution
Get 10 of test samples, accurately weighed, chopping, mixes, precision takes in right amount, puts in 100ml volumetric flask, adds acetonitrile to scale, put 45 ℃ of water-bath joltings timeazoamine ester is dissolved, on shaker, vibrate after 2min, then put into refrigerator freezing and filter immediately after 1 hour, precision measures the subsequent filtrate 1ml of putting to room temperature, puts in 10ml volumetric flask, with mobile phase, is diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that contains 0.05mg in every 1ml, as need testing solution
(3) assay: draw reference substance solution, need testing solution, each 10-20ul, injects high performance liquid chromatograph, records chromatogram, according to the peak area of chromatogram, calculates the content of timeazoamine ester in test sample,
Wherein, chromatographic condition is as follows: chromatographic column: the ODS-2HYPERSIL(250mm * 4.6mm of Thermo company, 5 μ m); Mobile phase is acetonitrile-0.04molL
-1sodium acetate and 0.01molL
-1ammonium acetate mixed solution is (containing 0.01molL
-1perfluorooctane sulfonate, pH=2.5, volume ratio 40: 60); Flow velocity 1.0mLmin
-1; Detection wavelength is 328nm; Sample size: 20 μ L; Column temperature: 40 ℃.
Method of the present invention obtains through screening, and screening process is as follows:
1 instrument and reagent
The U.S. Agilent1100 of Agilent company high performance liquid chromatograph: comprise the online degasser of Agilent1100G1322A, Agilent1100G1311A quaternary pump, Agilent1100G1313A automatic sampler, Agilent1100G1316A column oven, Agilent1100G1315B diode array detector (DAD);
Acetonitrile is chromatographically pure, and water is redistilled water, and it is pure that other reagent are domestic analysis;
Timeazoamine ester reference substance (normalization content is 99.8%) and sample (lot number: 090601,090602,090603) by Tianjin Tian Shili group chemical pharmacy Research Institute.
2. solution preparation
The preparation of 2.1 reference substance solution
It is appropriate that precision takes timeazoamine ester reference substance, dissolves and be diluted in every 1ml to contain tizolemide ester 0.05mg solution with mobile phase, obtains.
The preparation of 2.2 need testing solutions
A method: get 10 of test samples, accurately weighed, chopping, mix, precision takes in right amount (being approximately equivalent to timeazoamine ester 50mg), puts in 100ml volumetric flask, adds acetonitrile to scale, put ultrasonic 10min in ultrasonic cleaner, put into refrigerator freezing (18 ℃) and filter immediately after 1 hour, precision measures the subsequent filtrate 1ml of putting to room temperature, puts in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that approximately contains 0.05mg in every 1ml, as need testing solution.
B method: get 10 of test samples, accurately weighed, chopping, mix, precision takes in right amount (being approximately equivalent to timeazoamine ester 50mg), puts in 100ml volumetric flask, adds acetonitrile to scale, putting 45 ℃ of water-bath joltings dissolves timeazoamine ester, put into refrigerator freezing (18 ℃) and filter immediately after 1 hour, precision measures the subsequent filtrate 1ml of putting to room temperature, puts in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that approximately contains 0.05mg in every 1ml, as need testing solution.
C method: get 10 of test samples, accurately weighed, chopping, mix, precision takes in right amount (being approximately equivalent to timeazoamine ester 50mg), put in 100ml volumetric flask, add acetonitrile to scale, putting 45 ℃ of water-bath joltings dissolves timeazoamine ester, on shaker, vibrate after 2min, putting into refrigerator freezing (18 ℃) filters after 1 hour immediately again, precision measures the subsequent filtrate 1ml of putting to room temperature, put in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that approximately contains 0.05mg in every 1ml, as need testing solution.
3. chromatographic condition
Chromatographic column: the ODS-2HYPERSIL(250mm * 4.6mm of Thermo company, 5 μ m); Mobile phase is acetonitrile-0.04molL
-1sodium acetate and 0.01molL
-1ammonium acetate mixed solution is (containing 0.01molL
-1perfluorooctane sulfonate, pH=2.5, volume ratio 40: 60); Flow velocity 1.0mLmin
-1; Detection wavelength is 328nm; Sample size: 20 μ L; Column temperature: 40 ℃.
4, auxiliary material interference test
Auxiliary material blank solution: precision takes mixed accessories 0.45g (being equivalent to the auxiliary material amount containing timeazoamine ester 50mg), put in 10ml volumetric flask, add acetonitrile to scale, in 45 ℃ of water-baths, after thawing, after shaker vibration 2min, put into freezer compartment of refrigerator, freezing 1 hour, take out and filter, put to room temperature, precision measures this solution 1ml and is diluted to 10ml with mobile phase, is mixed with not containing the blank auxiliary material solution of timeazoamine ester, gets this solution sample introduction 20 μ l and records chromatogram.
Under above-mentioned chromatographic condition, the chromatogram of reference substance, test sample, auxiliary material blank solution is shown in accompanying drawing.Result shows, auxiliary material is without chromatogram absorption peak, noiseless to measuring.
5. the investigation of linear relationship
Precision takes timeazoamine ester reference substance 12.5mg, puts in 25ml measuring bottle, with acetonitrile, dissolves and is diluted to scale (0.5mg/ml).Precision measures above-mentioned solution 0.5,1,1.5,2,3ml, put in 10ml measuring bottle, add mobile phase and be diluted to scale, shake up, 0.025,0.05,0.075,0.1, the solution of 0.15mg/ml, get respectively each concentration sample solution sample introduction 20 μ l, by the chromatographic condition in " 3 ", measure, record chromatogram.Adopt least square method to carry out linear regression to the peak area of timeazoamine ester in chromatogram (A) and reference substance solution mass concentration (C), regression equation is: A=45624C+15.998, r=0.9999.Result shows that timeazoamine ester mass concentration is at 0.025～0.15mgmL
-1in scope, be good linear relation.
6. precision test
The accurate reference substance solution 20 μ l that draw, by the chromatographic condition continuous sample introduction in " 3 " 6 times, record chromatographic peak area.The RSD of result timeazoamine ester peak area is 0.04%, shows that the precision of instrument is good.
7. stability test:
Get with a need testing solution (090601 batch), respectively at 0,2,4,6,8,12,24h, by chromatographic condition in " 3 ", measure, record chromatographic peak peak area.The RSD of result timeazoamine ester peak area is 0.28%, result show need testing solution place 24 hours basicly stable.
8. replica test
Precision takes 6 parts of same batch samples (090601 batch), makes need testing solution, and measure by chromatographic condition sample introduction in " 3 " by " 2.2.3 " below method, and result timeazoamine ester labelled amount (n=6) is 98.45%; RSD is 1.89%.
9. recovery test:
9 parts of be labelled amount in prescription ratio preparation containing timeazoamine ester 80%, 100%, 120% analog samples, adopt respectively the method under " 2.2 " item to carry out pre-treatment to sample, obtain need testing solution, another accurate preparation is containing the reference substance solution of timeazoamine ester 0.05mg/ml, sample introduction, record chromatographic peak area, external standard method calculate recovery rate, the results are shown in Table 1.Average recovery rate (n=9) is 99.82%.
Table 1 recovery test result (n=9)
10. sample size is measured
According to the method C under " 3 " lower chromatographic condition and " 2.2 " item, content in 3 batch samples is measured, be the results are shown in Table 2.
Table 2 sample size measurement result (labelled amount %, n=3)
11. discuss
Detect the selection of wavelength: the present invention adopts UV spectrophotometer to carry out full wavelength scanner to need testing solution, and timeazoamine ester has absorption maximum near 328nm, therefore select 328nm as detecting wavelength.
The selection of chromatographic column and mobile phase: the mobile phase of original adoption, acetonitrile-0.04molL
-1sodium acetate and 0.01molL
-1ammonium acetate mixed solution is (containing 0.01molL
-1perfluorooctane sulfonate, pH=2.5, volume ratio 35: 65), found that, the appearance time of this product main peak is longer.Through test, the person of choosing acetonitrile-0.04molL
-1sodium acetate and 0.01molL
-1ammonium acetate mixed solution is (containing 0.01molL
-1perfluorooctane sulfonate, pH=2.5, volume ratio 40: 60) as mobile phase, and timeazoamine ester peak can obtain good separation, and the retention time of chromatographic peak advanceed in 20 minutes.
The selection of sample treatment: the matrix of suppository is insoluble to mobile phase, can not direct injected must pass through pre-treatment, the present invention has compared after ultrasonic extraction, heat fused after freezing processing and heat fused vibration 2min the preprocess methods such as freezing processing again, and the recovery is respectively 41.55%, 89.18%, 99.82%.Finally selected after heat fused vibration 2min again freezing processing for the assay of suppository.
Beneficial effect of the present invention is:
The focus that the present invention pays close attention to is, guarantees in the situation of suppository quality, and content that can convenient, fast and accurate mensuration timeazoamine ester suppository, has attempted multiple preprocess method, has set up reversed phase ion to high performance liquid chromatography.The method is simple, and favorable reproducibility can be used for said preparation assay.
Accompanying drawing explanation:
The HPLC chromatogram of Fig. 1 reference substance, wherein peak 1 is timeazoamine ester peak
The HPLC chromatogram of Fig. 2 test sample, wherein peak 1 is timeazoamine ester peak
The HPLC chromatogram of Fig. 3 auxiliary material blank solution
Embodiment:
Further illustrate by the following examples the present invention, but not as limitation of the present invention.
The preparation of embodiment 1 timeazoamine ester suppository
Prescription:
By recipe quantity, take timeazoamine ester stock and adjunct, heating and melting, stirs; The liquid preparing is injected to suppository mold tool, and liquid slightly overflows mould, cooling; Prune and overflow part, the demoulding, packs and get final product.
The detection of embodiment 2 timeazoamine ester suppositorys
(1) preparation of reference substance solution
It is appropriate that precision takes timeazoamine ester reference substance, dissolves and be diluted in every 1ml to contain tizolemide ester 0.05mg solution with mobile phase, obtains.
(2) preparation of need testing solution
Get 10 of test samples, accurately weighed, chopping, mix, precision takes in right amount (being approximately equivalent to timeazoamine ester 50mg), puts in 100ml volumetric flask, add acetonitrile to scale, put 45 ℃ of water-bath joltings timeazoamine ester is dissolved, on shaker, vibrate after 2min, then put into refrigerator freezing (18 ℃) and filter immediately after 1 hour, precision measures the subsequent filtrate 1ml of putting to room temperature, put in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that approximately contains 0.05mg in every 1ml, as need testing solution.
(3) assay: chromatographic condition, chromatographic column: the ODS-2HYPERSIL(250mm * 4.6mm of Thermo company, 5 μ m); Mobile phase is acetonitrile-0.04molL
-1sodium acetate and 0.01molL
-1ammonium acetate mixed solution is (containing 0.01molL
-1perfluorooctane sulfonate, pH=2.5, volume ratio 40: 60); Flow velocity 1.0mLmin
-1; Detection wavelength is 328nm; Sample size: 20 μ L; Column temperature: 40 ℃.Draw reference substance solution, need testing solution, each 10ul, injects high performance liquid chromatograph, records chromatogram, according to the peak area of chromatogram, calculates the content 99.45% of timeazoamine ester in test sample.
The detection of embodiment 3 timeazoamine ester suppositorys
(1) preparation of reference substance solution
It is appropriate that precision takes timeazoamine ester reference substance, dissolves and be diluted in every 1ml to contain tizolemide ester 0.025mg solution with mobile phase, obtains.
(2) preparation of need testing solution
Get 10 of test samples, accurately weighed, chopping, mix, precision takes in right amount (being approximately equivalent to timeazoamine ester 50mg), puts in 100ml volumetric flask, add acetonitrile to scale, put 45 ℃ of water-bath joltings timeazoamine ester is dissolved, on shaker, vibrate after 2min, then put into refrigerator freezing (18 ℃) and filter immediately after 1 hour, precision measures the subsequent filtrate 1ml of putting to room temperature, put in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that approximately contains 0.05mg in every 1ml, as need testing solution.
(3) assay: chromatographic condition, chromatographic column: the ODS-2HYPERSIL(250mm * 4.6mm of Thermo company, 5 μ m); Mobile phase is the mixed solution of acetonitrile-sodium acetate and ammonium acetate, volume ratio 35: 65, and wherein, the mixed solution of described sodium acetate and ammonium acetate contains 0.03molL
-1sodium acetate and 0.005molL
-1ammonium acetate, also contains 0.01molL in the mixed solution of sodium acetate and ammonium acetate
-1perfluorooctane sulfonate, its pH=2.5;
Flow velocity 0.5mLmin
-1;
Detection wavelength is 300nm;
Sample size: 10 μ L;
Column temperature: 30 ℃.
Draw reference substance solution, need testing solution, each 15ul, injects high performance liquid chromatograph, records chromatogram, according to the peak area of chromatogram, calculates the content 99.63% of timeazoamine ester in test sample.
The detection of embodiment 4 timeazoamine ester suppositorys
(1) preparation of reference substance solution
It is appropriate that precision takes timeazoamine ester reference substance, dissolves and be diluted in every 1ml to contain tizolemide ester 0.075mg solution with mobile phase, obtains.
(2) preparation of need testing solution
Get 10 of test samples, accurately weighed, chopping, mix, precision takes in right amount (being approximately equivalent to timeazoamine ester 50mg), puts in 100ml volumetric flask, add acetonitrile to scale, put 45 ℃ of water-bath joltings timeazoamine ester is dissolved, on shaker, vibrate after 2min, then put into refrigerator freezing (18 ℃) and filter immediately after 1 hour, precision measures the subsequent filtrate 1ml of putting to room temperature, put in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that approximately contains 0.05mg in every 1ml, as need testing solution.
(3) assay: chromatographic condition, chromatographic column: the ODS-2HYPERSIL(250mm * 4.6mm of Thermo company, 5 μ m); Mobile phase is the mixed solution of acetonitrile-sodium acetate and ammonium acetate, volume ratio 45: 55, and wherein, the mixed solution of the good ammonium acetate of described sodium acetate contains 0.05molL
-1sodium acetate and 0.02molL
-1ammonium acetate, also contains 0.01molL in the mixed solution of the good ammonium acetate of sodium acetate
-1perfluorooctane sulfonate, its pH=2.5;
Flow velocity 1.5mLmin
-1;
Detection wavelength is 350nm;
Sample size: 20 μ L;
Column temperature: 50 ℃.
Draw reference substance solution, need testing solution, each 20ul, injects high performance liquid chromatograph, records chromatogram, according to the peak area of chromatogram, calculates the content 99.22% of timeazoamine ester in test sample.
Claims (5)
1. by a method for RP-Ion Pair HPLC timeazoamine ester suppository content, it is characterized in that, comprise the steps:
(1) preparation of reference substance solution;
(2) preparation of need testing solution;
(3) assay: draw reference substance solution, need testing solution, each 10-20ul, injects high performance liquid chromatograph, records chromatogram, according to the peak area of chromatogram, calculates the content of timeazoamine ester in test sample;
Wherein, chromatographic condition is as follows: chromatographic column: octadecylsilane chemically bonded silica ODS-2HYPERSIL; Mobile phase is acetonitrile-sodium acetate and ammonium acetate mixed solution, wherein contains perfluorooctane sulfonate.
2. according to the method for claim 1, it is characterized in that, described in step (1), the preparation method of reference substance solution is as follows:
Take timeazoamine ester reference substance appropriate, with mobile phase, dissolve and be diluted to the solution that contains timeazoamine ester 0.025-0.075mg in every 1ml, obtain.
3. according to the method for claim 1, it is characterized in that, described in step (2), the preparation method of need testing solution is as follows:
Get 10, test sample timeazoamine ester bolt, accurately weighed, chopping, mix, precision takes in right amount, be equivalent to timeazoamine ester 50mg, put in 100ml volumetric flask, add acetonitrile to scale, putting 45 ℃ of water-bath joltings dissolves timeazoamine ester, on shaker, vibrate after 2min, putting into refrigerator freezing filters after 1 hour immediately again, precision measures the subsequent filtrate 1ml of putting to room temperature, put in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that contains 0.05mg timeazoamine ester in every 1ml, as need testing solution.
4. according to the method for claim 1, it is characterized in that, described chromatographic condition is as follows:
Chromatographic column: the ODS-2HYPERSIL(250mm * 4.6mm of Thermo company, 5 μ m);
Mobile phase is the mixed solution of acetonitrile-sodium acetate and ammonium acetate, volume ratio 35-45: 55-65, and wherein, the mixed solution of described sodium acetate and ammonium acetate contains 0.03-0.05molL
-1sodium acetate and 0.005-0.02molL
-1ammonium acetate, also contains 0.01molL in the mixed solution of sodium acetate and ammonium acetate
-1perfluorooctane sulfonate, its pH=2.5;
Flow velocity 0.5-1.5mLmin
-1;
Detection wavelength is 300-350nm;
Sample size: 10-20 μ L;
Column temperature: 30-50 ℃.
5. according to the method for claim 1, it is characterized in that, step is as follows:
(1) preparation of reference substance solution
It is appropriate that precision takes timeazoamine ester reference substance, dissolves and be diluted in every 1ml to contain tizolemide ester 0.05mg solution with mobile phase, obtain,
(2) preparation of need testing solution
Get 10 of test samples, accurately weighed, chopping, mixes, precision takes in right amount, be equivalent to timeazoamine ester 50mg, put in 100ml volumetric flask, add acetonitrile to scale, putting 45 ℃ of water-bath joltings dissolves timeazoamine ester, on shaker, vibrate after 2min, then put into refrigerator freezing and filter immediately after 1 hour, precision measures the subsequent filtrate 1ml of putting to room temperature, put in 10ml volumetric flask, with mobile phase, be diluted to scale, shake up, through 0.45 μ m miillpore filter, filter, get subsequent filtrate, make the solution that contains 0.05mg in every 1ml, as need testing solution
(3) assay: draw reference substance solution, need testing solution, each 10-20ul, injects high performance liquid chromatograph, records chromatogram, according to the peak area of chromatogram, calculates the content of timeazoamine ester in test sample,
Wherein, chromatographic condition is as follows: chromatographic column: the ODS-2HYPERSIL(250mm * 4.6mm of Thermo company, 5 μ m); Mobile phase is acetonitrile-0.04molL
-1sodium acetate and 0.01molL
-1ammonium acetate mixed solution is (containing 0.01molL
-1perfluorooctane sulfonate, pH=2.5, volume ratio 40: 60); Flow velocity 1.0mLmin
-1; Detection wavelength is 328nm; Sample size: 20 μ L; Column temperature: 40 ℃.
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