CN101126747B - Tumour cell proliferation imaging agent precursor content analysis method - Google Patents
Tumour cell proliferation imaging agent precursor content analysis method Download PDFInfo
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- CN101126747B CN101126747B CN 200710026141 CN200710026141A CN101126747B CN 101126747 B CN101126747 B CN 101126747B CN 200710026141 CN200710026141 CN 200710026141 CN 200710026141 A CN200710026141 A CN 200710026141A CN 101126747 B CN101126747 B CN 101126747B
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Abstract
The utility model relates to an analysis method for the content of a proliferation developer precursor in tumor cells, in particular to an analysis method for the content of (5'-o-(4,4'-dimethoxy-triphenylmethyl)-2'-deoxidation-3'-O-(4-nitro-benzenesulfonyl)-Beta-D-threonine furan pentose) (Noso-FLT), belonging to the apparatus analysis technical field. The utility model provides a measurement method for Noso-FLT content which is used by a powerful probe unit of a liquid phase chromatogram-evaporation light dispersion, displaying well the separation of the main composite peak and the impuritypeak, the linearity scope is 10 to 100ug/ml, the minimum test capacity is 5.0ng(S/N is more than or equal to 3), and the relative standard deviation of the test result is about 1.25%. The utility model adopts the probe unit of the liquidoid chromatogram evaporation light dispersion to the content of Noso-FLT and the content of the related impurities and has advantages of simple method and reliability.
Description
Technical field
A kind of content analysis method of tumour cell proliferation imaging agent precursor; be specifically related to that [5 '-O-(4; 4 '-dimethoxytrityl)-2 '-and deoxidation-3 '-O-(4-nitrobenzene sulfonyl)-β-D-threo form furan pentose base] content analysis method of thymidine, belong to the instrument analysis technology field.
Background technology
[5 '-O-(4,4 '-dimethoxytrityl)-2 '-deoxidation-3 '-O-(4-nitrobenzene sulfonyl)-β-D-threo form furan pentose base] thymidine (Noso-FLT) be a kind of tumour cell proliferation imaging agent 3 '-deoxidation-3 '-fluorine thymidine (
18F-FLT, the labelled precursor of 3 '-deoxy-3 '-fluorothymidine).Its chemical structural formula is as follows:
18It is stable that F-FLT has chemical property, in vivo can very fast decomposition, can be used to detect advantages such as cell proliferation.The propagation situation that reflects tumour cell by the activity of position emissron tomography PET video picture reflection thymidine kinase 1 (TK-1) indirectly helps that tumour is carried out good pernicious discriminating, curative effect assessment and prognosis and judges.Present how tame PET center applications
18F-FLT has carried out cell line research, zoopery and clinical application research extensively and profoundly to kinds of tumors.Numerous studies show that,
18F-FLT may be a kind of ratio clinical tumour PET medicine commonly used at present
18F-FDG (
18The F-deoxyglucose) the PET tracer agent that tumour-specific is higher, and may not have in early days that wound is estimated the oncotherapy reaction and the prognosis evaluation aspect has important value, be PET developer with application prospect.
In order to obtain high-quality PET tumour cell proliferation imaging agent
18F-FLT, in the process of preparation precursor Noso-FLT, must strict control impurity content.Can detect Noso-FLT with general UV-detector, but some are not had uv absorption or the more weak impurity of uv absorption, can't detect.The ultraviolet detection chromatogram is seen Fig. 1.
We adopt evaporative light-scattering detector (ELSD), after being characterized in that eluent enters detecting device, at first atomized by high pressure draught, the droplet that atomizing forms enters vaporization chamber (drift tube, drift tube), and moving phase and lower boiling component are evaporated, the droplet of remaining high boiling component enters scattering cell, be scattered when light beam passes scattering cell, scattered light is received by photoelectric tube and forms electric signal, and electric signal converts chromatogram digital signal---chromatogram to by amplifying circuit, analog digital.All enter into the material of scattering cell all can be detected, and response is only relevant with amount of substance, and comparing it with common detector differential detecting device does not have the refractive power parallax effect, has the advantage that baseline wander can not occur.Therefore, we can detect all impurity among the Noso-FLT with HPLC-ELSD, comprise the impurity of no uv absorption, and chromatogram is seen Fig. 2.Correlation technique is not seen bibliographical information.
Summary of the invention
The purpose of this invention is to provide a kind of Noso-FLT analysis on Content method, we have determined the inventive method after adopting high performance liquid chromatography-evaporative light-scattering (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 (HPLC-ELSD) that Noso-FLT is detected, chromatographic condition is defined as: instrument: WATERS 600 type high performance liquid chromatographs, WATERS 2420 evaporative light-scattering detector, chromatographic column Lichrospher C8 post, 5 μ m, 4.6 * 250mm, moving phase: methyl alcohol: water: trifluoroacetic acid (TFA) volume ratio is 90: 10: 0.1, also outgas flow velocity: 0.5mL/min before using through the organic filter membrane suction filtration of 0.45 μ m; The sampling volume of Noso-FLT sample: 5 μ L; Drift tube temperature: 45～60 ℃; N
2Pressure 25～35psi; Column temperature: room temperature.Precision takes by weighing the Noso-FLT reference substance of dry constant weight, is mixed with the solution that contains Noso-FLT 0.5mg among every 1mL with methyl alcohol, counts W
Contrast(W is a concentration, the mg/mL of unit, down together), precision takes by weighing sample, is mixed with methyl alcohol to contain the solution that tested Noso-FLT sample is controlled to be 0.5mg among every 1mL, counts W
Sample, measure the gained peak area and be respectively S
Contrast, S
Sample, the content that calculates Noso-FLT 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 Noso-FLT content with high performance liquid chromatography-evaporative light-scattering detector, show that the major component peak separates with impurity peaks well, the range of linearity is 10～100 μ g/mL, and minimum detectable activity is 5.0ng (S/N 〉=3).The relative standard deviation of gained testing result is about 1.25%.The present invention uses the liquid chromatography evaporative light-scattering detector to analyze content and the relative substance content of Noso-FLT, and method is easy, reliable.
Description of drawings
Fig. 1 Noso-FLT ultraviolet detection chromatogram
Fig. 2 Noso-FLT evaporative light-scattering detects chromatogram
Fig. 3 methanol solution chromatogram
Fig. 4 Noso-FLT is to chromatogram in the same old way
Fig. 5 Noso-FLT sample chromatogram figure
Fig. 6 Noso-FLT alkali destroys chromatogram
Fig. 7 Noso-FLT acid destroys chromatogram
Embodiment
The selection of embodiment 1 moving phase
Chromatographic column adopting C8 post, 5 μ m, 4.6 * 250mm is respectively with following moving phase test
(I) methanol-water (90: 10)
(J) methanol-water (80: 20)
(K) methanol-water (60: 40)
(L) methanol-water (50: 50)
(M) methanol-water-TFA (90: 10: 0.1)
(N) methanol-water-TFA (80: 20: 0.1)
(O) methanol-water-TFA (60: 40: 0.1)
(P) methanol-water-TFA (50: 50: 0.1)
With A～H is the chromatogram of moving phase, wherein between main peak and the impurity peaks suitable degree of separation is arranged in the E chromatogram, 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 45 ℃～60 ℃.
Embodiment 3 carrier gas (N
2) the pressure setting
Carrier gas (N
2) pressure selects 15,25,35, the 45psi test, the chromatogram effect was better when pressure was 25～35psi.
Therefore, chromatographic condition is defined as: instrument: WATERS 600 type high performance liquid chromatographs, WATERS2420 evaporative light-scattering detector.Chromatographic column Lichrospher C8 post, 5 μ m, 4.6 * 250mm, moving phase: methanol-water-TFA (90: 10: 0.1) also outgases through the organic filter membrane suction filtration of 0.45 μ m before using.Flow velocity: 0.5ml/min; Sampling volume: 10 μ l; The column temperature room temperature; 45 ℃～60 ℃ of ELSD drift tube temperatures, carrier gas (N
2) pressure is 25～35psi.
Embodiment 4 blank tests
Therefore this product dissolve with methanol has examined or check the influence of methyl alcohol to chromatographic behavior, is shown as the blank HPLC chromatogram of methyl alcohol, shows the solvent-free peak of methyl alcohol, noiseless to chromatographic behavior.
Embodiment 5 ranges of linearity and detectability test
It is an amount of accurately to take by weighing a certain amount of reference substance Noso-FLT, becomes 5mg/mL with dissolve with methanol, and sample introduction 2,5,10,15,20 μ L map to sample size with integral area respectively.The gained regression equation is y=10352x-11879, R
2=0.9958.The range of linearity is 10～100 μ g/mL.It is an amount of to get reference substance, is made among every 1ml with methyl alcohol to contain 0.5 * 10
-6The solution of mg is as need testing solution.Minimum detectable activity is 5.0ng (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 1.25% according to calculated by peak area gained relative standard deviation.
Embodiment 7 sample sizes are measured
The Noso-FLT 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 Noso-FLT 50 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 50 μ g (W of tested Noso-FLT 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 Noso-FLT content in the external standard method calculation sample be
(S
Sample/ S
Contrast) * (W
Contrast/ W
Sample) * 100%.
The sample determination result
The sample determination result
It is an amount of to get this sample, and accurate the title decides, and is mixed with the solution that contains Noso-FLT 5mg among every 1mL, solution in contrast with methyl alcohol.In addition with methyl alcohol be mixed with contain Noso-FLT 0.05mg among every 1mL solution as need testing solution.Get contrast solution 5 μ L injecting chromatographs, according to high performance liquid chromatography (2005 editions two appendix V D of Chinese Pharmacopoeia) test, pillar C8 post, moving phase: methanol-water-TFA (90: 10: 0.1), 45-60 ℃ of ELSD drift tube temperature, carrier gas (N
2) pressure is 25-35psi.Regulate instrumental sensitivity, the peak height that makes the Noso-FLT 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 in this sample, 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)
- One kind [5 '-O-(4,4 '-dimethoxytrityl)-2 '-and deoxidation-3 '-O-(4-nitrobenzene sulfonyl)-β-D-threo form furan pentose base] thymidine analysis on Content method, it is characterized in that adopting high performance liquid chromatography-evaporative light-scattering that it is detected, this compound is called for short Noso-FLT, chromatographic condition is defined as: instrument: WATERS 600 type high performance liquid chromatographs, WATERS 2420 evaporative light-scattering detector, chromatographic column Lichrospher C8 post, 5 μ m, 4.6 * 250mm, moving phase: methyl alcohol: water: the trifluoroacetic acid volume ratio is 90: 10: 0.1, also outgases flow velocity: 0.5mL/min through the organic filter membrane suction filtration of 0.45 μ m before using; The sampling volume of Noso-FLT sample: 5 μ L; Drift tube temperature: 45～60 ℃; N 2Pressure 25～35psi; Column temperature: room temperature, precision take by weighing the Noso-FLT reference substance of dry constant weight, are mixed with the solution that contains Noso-FLT0.5mg among every 1mL with methyl alcohol, count W Contrast, precision takes by weighing sample, is mixed with methyl alcohol to contain the solution that tested Noso-FLT sample is controlled to be 0.5mg among every 1mL, counts W Sample, measure the gained peak area and be respectively S Contrast, S Sample, the content that calculates Noso-FLT in the sample by external standard method is:(S Sample/ S Contrast) * (W Contrast/ W Sample) * 100%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1595147A (en) * | 2004-07-02 | 2005-03-16 | 桂林三金药业股份有限公司 | Quality control method for Sanjin preparation |
| CN1786706A (en) * | 2004-12-10 | 2006-06-14 | 天津天士力制药股份有限公司 | Cyclovirobuxine D raw medicine and method for determining its content in preparation by chromatography |
| CN1904607A (en) * | 2006-07-31 | 2007-01-31 | 江苏省原子医学研究所 | Analysis method of mercapto amine tropine content |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1595147A (en) * | 2004-07-02 | 2005-03-16 | 桂林三金药业股份有限公司 | Quality control method for Sanjin preparation |
| CN1786706A (en) * | 2004-12-10 | 2006-06-14 | 天津天士力制药股份有限公司 | Cyclovirobuxine D raw medicine and method for determining its content in preparation by chromatography |
| CN1904607A (en) * | 2006-07-31 | 2007-01-31 | 江苏省原子医学研究所 | Analysis method of mercapto amine tropine content |
Non-Patent Citations (4)
| Title |
|---|
| 宋燕玲 |
| 席志芳.高效液相色谱-蒸发光散射检测法测定磷霉素钙胶囊含量.中国抗生素杂志31 4.2006,31(4),S1-S2. |
| 衷红梅 |
| 衷红梅;宋燕玲;席志芳.高效液相色谱-蒸发光散射检测法测定磷霉素钙胶囊含量.中国抗生素杂志31 4.2006,31(4),S1-S2. * |
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Owner name: WUXI JIANGYUAN AMS MOLECULAR NUCLEAR MEDICINE RESE Free format text: FORMER OWNER: JIANGSU PROV. INST. OF ATOMIC MEDICINE Effective date: 20111226 |
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Effective date of registration: 20111226 Address after: 214063 Jiangsu province Wuxi Qin new technology park Qian Rong Lu No. 1013 -A16 Patentee after: Wuxi Jiangyuan Andike Molecular Nuclear Medical Development Research Institute Address before: 214063 Jiangsu city of Wuxi province Qian Rong Lu No. 20 Patentee before: Jiangsu Prov. Inst. of Atomic Medicine |
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