CN1904607A - Analysis method of mercapto amine tropine content - Google Patents
Analysis method of mercapto amine tropine content Download PDFInfo
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- CN1904607A CN1904607A CNA2006100408976A CN200610040897A CN1904607A CN 1904607 A CN1904607 A CN 1904607A CN A2006100408976 A CNA2006100408976 A CN A2006100408976A CN 200610040897 A CN200610040897 A CN 200610040897A CN 1904607 A CN1904607 A CN 1904607A
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Abstract
The invention relates to a content analysis method for sulfhydryl-amine tropine. It uses high efficiency liquid phase chromatogram-vaporizing light scattering detector to test sulfhydryl-amine tropine. The devices that are used for chromatographic analyzing are WATERS 600 high efficiency liquid phase chromatograph, WATERS 2420 vaporizing light scattering detector, and the chromatographic column of Hypersil C8 column, 5um, 4.6*250mm. The results shows that it has good separation of the main constituents peak and the impurity peak, the linear range is 12.5-50ug/mL, and the minimum detecting quality is 3.0ng (S/N>=3), and that has about 3.6% error compared to relative standard error. The invention could test content and impurity of sulfhydryl-amine tropine, and it is simple and convenient.
Description
Technical field
A kind of analytical approach of mercapto amine tropine content belongs to the instrument analysis technology field.
Background technology
Mercapto amine tropine (TRODAT-1), the chinesization formal name used at school: 2 β-[N, N '-two (2-mercapto ethyl) ethylene diamin(e) base] methyl-3 β-(4-chlorphenyl) tropane, its chemical structural formula is as follows:
Mercapto amine tropine through technetium [
99mTc] product of mark be technetium [
99mTc] mercapto amine tropine (
99mTc-TRODAT-1), chinesization formal name used at school: technetium [
99mTc]-2 β-[N, N '-two (2-mercapto ethyl) ethylene diamin(e) base] methyl-3 β-(4-chlorphenyl) tropane, this compound is the novel dopamine transporter imaging agent of succeeding in developing in recent years, be in the world first successfully be used for clinical research technetium [
99mTc] the brain acceptor class developer of mark, technetium [
99mTc] mark radiopharmaceutical field, nuclear pharmacy field, cerebral receptor imaging field all be a breakthrough.A large amount of preclinical pharmacology both domestic and external, toxicological study and clinical research confirmation, technetium [
99mTc] mercapto amine tropine is the brain imaging agent that has clinical value, and the diagnosis of Parkinson's (PD) is had clinical value.
99mTc-TRODAT-1 has carried out vast amount of clinical abroad as the atomic disposable diagnostic medication of a kind of consumption, finds the diagnosis of PD is had suitable sensitivity and accuracy rate.This medicine is carrying out the clinical research of three phases in Europe by GE company; And the nuclear energy research institute in China Taiwan Province has obtained the new drug permission (the Drug System R00023 No. of Wei administration) in Taiwan Province in June, 2005; The domestic kind new medicine declaration work that just carrying out, wherein technetium [
99mTc] the content analysis of TRODAT-1 before the mark is the key of medicine box quality control.Owing to lack chromophoric group in the TRODAT-1 structure, general UV-detector is difficult to detect, and does not have reported in literature to cross its detection method of content.Evaporative light-scattering detector (ELSD) is atomized into fine droplet with the post eluent, and moving phase was evaporated after stream of liquid droplets was crossed the warm up drift pipe.Laser is by unevaporated sample particle scattering, and scattered light detects with silicon photoelectric diode.The detection of ELSD does not rely on the optical characteristics of sample, but any involatile constituent in the detection by quantitative sample, so we adopt HPLC-ELSD to detect the content of TRODAT-1.
Summary of the invention
The analytical approach that the purpose of this invention is to provide a kind of mercapto amine tropine content because of there not being chromophoric group in the mercapto amine tropine molecule, therefore can not be detected with common UV-detector.We have determined the inventive method after adopting high performance liquid chromatography-evaporative light-scattering detector (HPLC-ELSD) that chromatographic behavior has been carried out a series of investigation.
Technical scheme of the present invention: adopt high performance liquid chromatography-evaporative light-scattering detector that mercapto amine tropine is detected, chromatographic condition is defined as: instrument: WATERS 600 type high performance liquid chromatographs, the WATERS2420 evaporative light-scattering detector, chromatographic column Hypersil C8 post, 5 μ m, 4.6 * 250mm, moving phase: methyl alcohol-acetonitrile-water-trifluoroacetic acid (TFA) volume ratio (80: 10: 10: 0.1), also outgas flow velocity: 1.0ml/min before using through the organic filter membrane suction filtration of 0.45 μ m; The sampling volume of mercapto amine tropine sample: 5 μ l; The column temperature room temperature; 60 ℃～80 ℃ of evaporative light-scattering detector drift tube temperatures, carrier gas (N
2) pressure is 0.24MPa (35psi).Precision takes by weighing the mercapto amine tropine reference substance of dry constant weight, makes the solution that contains mercapto amine tropine 250 μ g among every 1ml with methyl alcohol, counts W
Contrast, precision takes by weighing sample, is made into to contain the solution that tested mercapto amine tropine sample is controlled to be 250 μ g among every 1ml, counts W
Sample, measure the gained peak area and be respectively S
Contrast, S
Sample, the content that calculates mercapto amine tropine in the sample by external standard method is:
(S
Sample/ S
Contrast) * (W
Contrast/ W
Sample) * 100%.
Beneficial effect of the present invention: the invention provides a kind of method that detects mercapto amine tropine content with high performance liquid chromatography-evaporative light-scattering detector (HPLC-ELSD), show that the major component peak separates with impurity peaks well, the range of linearity is 12.5～50 μ g/mL, and minimum detectable activity is 3.0ng (S/N 〉=3).The relative standard deviation of gained testing result is about 3.6%.The peak area sum of each impurity peaks is no more than 25% of reference substance total peak area in the need testing solution.The present invention analyzes, and method is easy, reliable.
Description of drawings
Fig. 1 mercapto amine tropine is TRODAT-1 with the peak of the separation graph retention time 4.906 of synthesizing two preceding intermediates, and two other peak is an intermediate.
The HPLC chromatogram of Fig. 2 methyl alcohol blank
The HPLC chromatogram of the methanol solution of two intermediates of Fig. 3
The HPLC chromatogram of Fig. 4 TRODAT-1 methanol solution
The HPLC chromatogram of Fig. 5 alkali failure test
The HPLC chromatogram of Fig. 6 acid failure test
Embodiment
The selection of embodiment 1 moving phase
The reaction equation of preparation mercapto amine tropine is:
Because compound 9 (2 β-[N-[2-[S-(4-methoxy-benzyl) sulfenyl] ethyl]-N-[[N-[2-[S-(4-methoxy-benzyl) sulfenyl] ethyl] amino] the formyl methyl] amino methyl]-3 β-(4-chlorphenyl) tropane) and compound 10 (2 β-[N-[2-[S-(4-methoxy-benzyl) sulfenyl] ethyl]-N-[2-[N-[2-[S-(4-methoxy-benzyl) sulfenyl] ethyl] amino] ethyl] amino methyl]-3 β-(4-chlorphenyl) tropane) be the synthetic intermediate of TRODAT-1; Thereby to investigate TRODAT-1 and their separation case.Be moving phase with following solution respectively, (4.6 * 150mm) test for Symmetry, 5 μ m with the C8 post.
(A) methanol-water (80: 20)
(B) methanol-water (60: 40)
(C) acetonitrile-water (80: 20)
(D) acetonitrile-water (60: 40)
(E) methanol-water-TFA (80: 20: 0.1)
(F) acetonitrile-water-TFA (80: 20: 0.1)
(G) methyl alcohol-acetonitrile-water-TFA is (80: 10: 10: 0.1)
Be that main peak and impurity peaks are overlapping in the chromatogram of moving phase with A, B, C, D; Be that the degree of separation of main peak and impurity peaks is less in the chromatogram of moving phase with E, F; Be that main peak well separates with middle physical efficiency in the chromatogram of moving phase with G, and the retention time of main peak is proper, as shown in Figure 1.
The setting of embodiment 2 drift tube temperatures
Drift tube temperature is made as 40 ℃～100 ℃ respectively tests, the result shows that chromatographic resolution was better when drift tube temperature was 60 ℃～80 ℃.Therefore, chromatographic condition is defined as: instrument: WATERS 600 type high performance liquid chromatographs, WATERS 2420 evaporative light-scattering detector.Chromatographic column Hypersil C8 post, 5 μ m, 4.6 * 250mm, moving phase: methyl alcohol-acetonitrile-water-TFA (80: 10: 10: 0.1), also outgas flow velocity: 1.0ml/min through the organic filter membrane suction filtration of 0.45 μ m before using; Sampling volume: 5 μ l; The column temperature room temperature; 60 ℃～80 ℃ of ELSD drift tube temperatures, carrier gas (N
2) pressure is 35psi.
Embodiment 3 carrier gas (N
2) flow set
Carrier gas (N
2) pressure selects 15,25,35, the 45psi test, the chromatogram effect was better when pressure was 35psi.
The embodiment 4 synthetic preceding intermediates and the separation test of major component
Therefore this product dissolve with methanol has examined or check the influence of methyl alcohol to chromatographic behavior, and Fig. 2 is shown as the blank HPLC chromatogram of methyl alcohol, shows that methyl alcohol is noiseless to chromatographic behavior.Fig. 3 is two intermediates (compounds 9 and 10 in the synthetic route, 2 β-[N-[2-[S-(4-methoxy-benzyl) sulfenyl] ethyl]-N-[[N-[2-[S-(4-methoxy-benzyl) sulfenyl] ethyl] amino] the formyl methyl] amino methyl]-3 β-(4-chlorphenyl) tropane (9), 2 β-[N-[2-[S-(4-methoxy-benzyl) sulfenyl] ethyl]-N-[2-[N-[2-[S-(4-methoxy-benzyl) sulfenyl] ethyl] amino] ethyl] amino methyl]-3 β-(4-chlorphenyl) tropane (10) and methanol solution HPLC chromatogram, Fig. 4 shows the TRODAT-1 chromatogram, shows that the major component peak separates well with impurity peaks.
It is an amount of accurately to take by weighing a certain amount of reference substance TRODAT-1, becomes 2.5mg/ml with dissolve with methanol, and sample introduction 5,10,15,20 μ l map to sample size with integral area respectively.The gained regression equation is y=12710x-12342, R
2=0.9988.The range of linearity is 12.5～50 μ g/mL.It is an amount of to get reference substance, is made among every 1ml with methyl alcohol to contain 6 * 10
-6The solution of mg is as need testing solution.Minimum detectable activity is 3.0ng (S/N 〉=3).
The test of embodiment 6 precision
Test sample under the chromatographic condition of determining on the same day in 6 mensuration of continuous sample introduction, be about 3.6% according to calculated by peak area gained relative standard deviation.
Embodiment 7 sample sizes are measured
The TRODAT-1 reference substance (prepared by this) of getting dry constant weight is an amount of, and accurate the title decides, and makes with methyl alcohol to contain TRODAT-1 250 μ g (W among every 1ml
Contrast) solution, solution in contrast.It is an amount of that other takes by weighing sample, is made into to contain the about 250 μ g (W of tested TRODAT-1 sample among every 1ml
Sample) solution as need testing solution.By getting 5 μ l injecting chromatographs under the above-mentioned chromatographic condition respectively, to measure, the gained peak area is respectively S contrast, S sample, by TRODAT-1 content in the external standard method calculation sample is
(S
Sample/ S
Contrast) * (W
Contrast/ W
Sample) * 100%.
Embodiment 8 related substance inspection technique and measurement results
It is an amount of to get this product, and accurate the title decides, and makes the solution that contains TRODAT-1 5mg among every 1ml, solution in contrast with methyl alcohol.Other be made into contain TRODAT-1 0.05mg among every 1ml solution as need testing solution.Get contrast solution 5 μ l injecting chromatographs, test according to high performance liquid chromatography (2005 editions two appendix V D of Chinese Pharmacopoeia), with the bonded silica gel is filling agent, moving phase: and methyl alcohol-acetonitrile-water-TFA (80: 10: 10: 0.1), 80 ℃ of ELSD drift tube temperatures, carrier gas (N
2) pressure is 35psi.Regulate instrumental sensitivity, the peak height that makes the TRODAT-1 peak is 15% ~ 30% of a registering instrument full scale.Measure need testing solution 5 μ l injecting chromatographs again, the record chromatogram is to 4 times of major component peak retention time.Calculate by area normalization method, the peak area sum of each impurity peaks is no more than 25% of reference substance total peak area in the need testing solution.
Failure test is carried out in embodiment 9 method specificity researchs
Get two parts of this product, add the 6mol/L NaOH solution dissolving of (1) 1ml respectively, the 6mol/L HCl solution dissolving of (2) 1ml is put boiling water bath respectively and was added heat damage 1 hour.Transfer pH to neutral respectively, evaporated under reduced pressure is used dissolve with methanol, and dilution is tested under above-mentioned chromatographic condition respectively.
The result shows; Under alkali, sour failure condition, increase, and separate well with main peak through the efficient liquid phase chromatographic analysis impurity peaks.This shows that this chromatogram system specificity is better, can detect various impurity.
Claims (1)
1. the analytical approach of a mercapto amine tropine content, it is characterized in that adopting high performance liquid chromatography-evaporative light-scattering detector that the mercapto amine tropine sample is detected, chromatographic condition is defined as: instrument: WATERS 600 type high performance liquid chromatographs, WATERS 2420 evaporative light-scattering detector, chromatographic column Hypersil C8 post, 5 μ m, 4.6 * 250mm, moving phase: methyl alcohol-acetonitrile-water-trifluoroacetic acid volume ratio is 80: 10: 10: 0.1, also outgas flow velocity: 1.0ml/min before using through the organic filter membrane suction filtration of 0.45 μ m; The sampling volume of mercapto amine tropine: 5 μ l; The column temperature room temperature; 60 ℃～80 ℃ of evaporative light-scattering detector drift tube temperatures, carrier gas N
2Pressure is 0.24MPa; Precision takes by weighing the mercapto amine tropine reference substance of dry constant weight, makes the solution that contains mercapto amine tropine 250 μ g among every 1ml with methyl alcohol, counts W
Contrast, precision takes by weighing sample, is made into to contain the solution that tested mercapto amine tropine sample is controlled to be 250 μ g among every 1ml, counts W
Sample, measure the gained peak area and be respectively S
Contrast, S
Sample, the content that calculates mercapto amine tropine in the sample by external standard method is: (S
Sample/ S
Contrast) * (W
Contrast/ W
Sample) * 100%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101126747B (en) * | 2007-08-16 | 2010-08-11 | 江苏省原子医学研究所 | Tumour cell proliferation imaging agent precursor content analysis method |
| CN101135676B (en) * | 2007-07-23 | 2010-08-11 | 江苏省原子医学研究所 | Boc protected thymidine derivates BOC-FLT content analysis method |
| CN101398414B (en) * | 2008-10-15 | 2011-07-20 | 上海市公安局刑事侦查总队 | Method for qualitatively screening 242 kinds of compounds by liquid phase chromatography-mass spectra at the same times |
| CN102353674A (en) * | 2011-06-27 | 2012-02-15 | 天津市新冠制药有限公司 | Analytical method for measuring capecitabine content with non-aqueous titration method |
| CN101320014B (en) * | 2008-07-01 | 2012-11-21 | 江苏省原子医学研究所 | Analytical method for measuring raw medicine content of thiohydroxylamine tropine by non-aqueous titrimetry |
| TWI577990B (en) * | 2015-09-25 | 2017-04-11 | 行政院原子能委員會核能研究所 | High performance liquid chromatography method for analyzing imaging agent, precursor of imaging agent, or intermediate of imaging agent |
| CN110646540A (en) * | 2019-09-30 | 2020-01-03 | 武汉嘉诺康医药技术有限公司 | HPLC-ELSD detection method for Retamolin starting material and intermediate |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI229187B (en) * | 2000-12-01 | 2005-03-11 | Iner Ae | A novel method for determination of radiochemical purity of technetium-99m-TRODAT-1 |
| CN1314456C (en) * | 2005-09-27 | 2007-05-09 | 江苏省原子医学研究所 | Medicament box of 2beta-[N,N'-di(2-mercaptoethyl) ethylene diamine] methyl-3 beta-(4- chlorphenyl) tropane |
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2006
- 2006-07-31 CN CNB2006100408976A patent/CN100368805C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101135676B (en) * | 2007-07-23 | 2010-08-11 | 江苏省原子医学研究所 | Boc protected thymidine derivates BOC-FLT content analysis method |
| CN101126747B (en) * | 2007-08-16 | 2010-08-11 | 江苏省原子医学研究所 | Tumour cell proliferation imaging agent precursor content analysis method |
| CN101320014B (en) * | 2008-07-01 | 2012-11-21 | 江苏省原子医学研究所 | Analytical method for measuring raw medicine content of thiohydroxylamine tropine by non-aqueous titrimetry |
| CN101398414B (en) * | 2008-10-15 | 2011-07-20 | 上海市公安局刑事侦查总队 | Method for qualitatively screening 242 kinds of compounds by liquid phase chromatography-mass spectra at the same times |
| CN102353674A (en) * | 2011-06-27 | 2012-02-15 | 天津市新冠制药有限公司 | Analytical method for measuring capecitabine content with non-aqueous titration method |
| TWI577990B (en) * | 2015-09-25 | 2017-04-11 | 行政院原子能委員會核能研究所 | High performance liquid chromatography method for analyzing imaging agent, precursor of imaging agent, or intermediate of imaging agent |
| CN110646540A (en) * | 2019-09-30 | 2020-01-03 | 武汉嘉诺康医药技术有限公司 | HPLC-ELSD detection method for Retamolin starting material and intermediate |
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| CN100368805C (en) | 2008-02-13 |
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