CN103645254A

CN103645254A - Method for analyzing content of A beta plaque developer precursor AV45

Info

Publication number: CN103645254A
Application number: CN201310614013.3A
Authority: CN
Inventors: 周杏琴; 张建康; 毛师师; 张荣军
Original assignee: Jiangsu Institute of Nuclear Medicine
Current assignee: Jiangsu Institute of Nuclear Medicine
Priority date: 2013-11-28
Filing date: 2013-11-28
Publication date: 2014-03-19
Anticipated expiration: 2033-11-28
Also published as: CN103645254B

Abstract

The invention relates to a method for analyzing the content of an A beta plaque developer precursor AV45, belonging to the technical field of apparatus analysis. An 18F-AV45 is capable of identifying an A beta plaque pathological state in the brain of an AD (Alzheimer Disease) patient in vivo, is used for visualized and quantitative research of an amyloid protein deposition process of a human body, and moves towards the clinical. A high-purity precursor AV45 needs to be obtained if a higher radiochemical purity PET (Positron Emission Tomography) developer 18F-AV45 is obtained. According to the method, the chromatography condition is as follows: a WATERS 600 type high performance liquid chromatograph, a 2487 ultraviolet detector, a chromatographic column Inertsil C18 column, 5 micrometers and 4.6*250mm; a flowing phase is as follows: a ratio v/v of 50mM disodium hydrogen phosphate to acetonitrile is 15-85, and 0.01 percent triethylamine is added; the flowing speed is 0.8mL/min; the AV45 sample feeding volume is 10mu L; the column temperature is the room temperature; and the detection wavelength is 325nm. According to the method, phosphate is used as a buffering agent, and the triethylamine is added, so that the trailing phenomenon is improved, and the peak symmetry is better. Compared with a literature method, the method has the advantages that the number of theoretical plates and the separation degree are greatly improved, and all impurities are separated well. Various impurities can be effectively detected by using 325nm detection.

Description

The content analysis method of a kind of A beta plaque imaging agent precursor AV45

Technical field

A content analysis method of A beta plaque imaging agent precursor AV45, belongs to instrument analysis technology field.

Background technology

Along with society enters aging, China's elderly population are more and more, the incidence of disease of Alzheimer disease (AD) rises year by year, current Chinese AD patient approximately has more than 5,000,000 people, become one of principal disease threatening quality of life in old age, bring huge financial burden to society and family, huge pressure and challenge will be born for this reason by following Chinese society.How effectively preventing and treating AD disease, improving old age quality of life is the very urgent key subjects that researcher is faced.At present, the pathogenesis of AD is still very not clear, and amyloid-beta (β-amyloid protein, A β) is assembled the main pathological characters that is considered to AD.Clinical manifestation is cognition dysfunction and being losing one's memory, and then causes human communication disorders, and self care ability is lost, and has various neuropsychic symptoms and behavior disorder.Early detection A beta plaque is assembled, and carries out anti-A beta plaque curative effect monitoring, is the effective measures of control AD.

Along with molecular imaging development, utilize single photon emission computerized tomography technology (SPECT) or PET (positron emission tomography) scanner (PET), adopt special developer and the A beta plaque specific binding in brain tissue, showing intuitively A beta plaque distribution situation in brain, is the international state-of-the-art molecular image technology of current early diagnosis AD.Therefore, develop special A beta plaque developer significant for early diagnosis and the therapeutic evaluation of AD.

¹⁸the positron emission fault with good clinical value (PET) the video picture reagent of F-AV45 Shi You U.S. Avid company development, is used for clinical by FDA approval at present. ¹⁸f-AV45 has good affinity to A beta plaque, can specific binding A beta plaque, at AD patient's cortical layer, there is remarkable delay, and AD patient and cognitive healthy volunteer can be significantly distinguished in PET video picture.A series of clinical before and clinical research show ¹⁸f-AV45 can differentiate A beta plaque pathological state in patient's AD brain at body, at present for the visual and quantitative examination of amyloid beta deposition process in human body, is about to move towards clinical.

¹⁸f-AV45(compound 3) be by labelled precursor (E)-2-(2-(2-(2-tolysulfonyl oxygen base oxethyl) ethoxy) ethoxy)-5-(4-tertbutyloxycarbonyl methylamino styrene) pyridine (compound 1) warp ¹⁸f mark is prepared from.

Obtain the pure higher PET developer of putting ¹⁸f-AV45, except control mark condition, another crucial part is the strict content of controlling precursor impurity in complex sign precursor, comprises the content of AV45 reaction intermediate (compound 2, hereinafter to be referred as intermediate), obtains highly purified labelled precursor AV45.

The HPLC method of bibliographical information adopts acetonitrile: Ammoniom-Acetate (85:15) is mobile phase, and peak type exists conditions of streaking, and peak is wider, can not impurity is effectively separated.This law adopts phosphate to make buffering agent, and adopts interpolation triethylamine that conditions of streaking is improved, and peak symmetry is better.Compare with literature method, theoretical cam curve and degree of separation are greatly improved, and each impurity is better separated.Adopting 325nm to detect can effectively be detected each impurity.

Summary of the invention

The object of the invention is to set up the content of high effective liquid chromatography for measuring A beta plaque imaging agent precursor AV45 and the content of related substance, for the quality control of medicine box provides effective analysis means, for clinical application provides quality control foundation, correlative study has no report.

Technical scheme of the present invention: the content analysis method of a kind of A beta plaque imaging agent precursor AV45, chromatographic condition: instrument: WATERS 600 type high performance liquid chromatographs, 2487 UV-detector, chromatographic column Inertsil C18 post, 5 μ m, 4.6 * 250mm; Mobile phase: 50mM Lin acid hydrogen Er Na ︰ acetonitrile volume ratio=15 ︰ 85, adds triethylamine v/v 0.01%, also degassed through the organic filter membrane suction filtration of 0.45 μ m before using; Flow velocity: 0.8mL/min; The sampling volume of AV45 sample: 10 μ L; Column temperature: room temperature; Detect wavelength: 325nm.

Beneficial effect of the present invention: this law adopts phosphate to make buffering agent, and adopt interpolation triethylamine that conditions of streaking is improved, peak symmetry is better.Compare with literature method, theoretical cam curve and degree of separation are greatly improved, and each impurity is better separated.Adopting 325nm to detect can effectively be detected each impurity.

Accompanying drawing explanation

Fig. 1 mobile phase acetonitrile: ammonium acetate system (85:15), 1, the HPLC chromatogram (Fig. 1-b) of precursor AV45,2, intermediate HPLC chromatogram (Fig. 1-a);

Fig. 2 dipotassium hydrogen phosphate system intermediate HPLC chromatogram, a: acetonitrile: 25mM dipotassium hydrogen phosphate (85:15), b: acetonitrile: 25mM dipotassium hydrogen phosphate (85:15) is containing 0.01% triethylamine;

Fig. 3 25mM sodium hydrogen phosphate system intermediate HPLC chromatogram, a: acetonitrile: 25mM sodium hydrogen phosphate (85:15), b: acetonitrile: 25mM sodium hydrogen phosphate (85:15) is containing 0.01% triethylamine;

Fig. 4 mobile phase acetonitrile: 50mM sodium dihydrogen phosphate system (85:15) is containing 0.01% triethylamine, 1, the HPLC chromatogram (Fig. 4-b) of precursor AV45,2, intermediate HPLC chromatogram (Fig. 4-a);

Fig. 5 AV45 (a) and intermediate (b) ultraviolet scanning spectrum figure;

Fig. 6 AV45 places the HPLC chromatogram after 3d, a: acetonitrile; B: ethanol; C: methyl alcohol;

Fig. 7 AV45 HPLC chromatogram, a: reference substance; B: sample.

Embodiment

Optimize chromatography condition:

The selection of mobile phase

Adopt WATERS 600 type high performance liquid chromatographs, 2487 UV-detector, chromatographic column Inertsil C18 post, 5 μ m, 4.6 * 250mm, respectively with following mobile phase test

(1) acetonitrile-Ammoniom-Acetate system

Adopting literature method Yi Jing ︰ ammonium acetate (85 ︰ 15) system is mobile phase, and results peaks type is wider, and has conditions of streaking.Theoretical cam curve is lower, is respectively 2600(2, intermediate), 5200(1, product).See Fig. 1.

(2) acetonitrile-dipotassium hydrogen phosphate system

A:25mM Lin acid hydrogen Er Jia ︰ acetonitrile=15 ︰ 85; B:25mM Lin acid hydrogen Er Jia ︰ acetonitrile=15 ︰ 85, adds 0.01% triethylamine.See Fig. 2.

(3) acetonitrile-sodium hydrogen phosphate system,

A:25mM Lin acid hydrogen Er Na ︰ acetonitrile=15 ︰ 85; B:25mM Lin acid hydrogen Er Na ︰ acetonitrile=15 ︰ 85, adds 0.01% triethylamine.See Fig. 3.

We adopt literature method, mobile phase acetonitrile: ammonium acetate system, results peaks type is wider, and has conditions of streaking.Theoretical cam curve is lower, is respectively 2600(2, intermediate), 5200(1, product).Consider in structure containing BOC blocking group (tertbutyloxycarbonyl), and end OTS has certain hydrophobicity, we adopt buffer system experiment.Adopt 25mM potassium dihydrogen phosphate, peak is still wider, and theoretical cam curve only has 700, use 25mM sodium dihydrogen phosphate system instead, peak type is sharpened, add triethylamine after theoretical cam curve from 600, be increased to 1600, sodium dihydrogen phosphate system is better than potassium dihydrogen phosphate system, but impurity peaks is failed effectively separately.From Fig. 3-b, can find out that triethylamine can improve the conditions of streaking at peak.

We have investigated the impact of ionic strength on degree of separation.The sodium dihydrogen phosphate buffer of preparation 15,25,35,45,50,65mM, mixes with 15 ︰ 85 volume ratios with acetonitrile respectively, adds triethylamine 0.01%, and mixed solvent is mobile phase sample introduction.Ionic strength on the impact of degree of separation and theoretical cam curve in Table 1.Final 50mM Lin acid dihydride Na ︰ acetonitrile (v/v) 15 ︰ 85 that select add triethylamine 0.01% for mobile phase, chromatogram see Fig. 4 (2, intermediate, 1, product), it is separated preferably that intermediate and impurity peaks, product and impurity peaks all have, and intermediate and product theoretical cam curve are all over 15000.

The impact of table 1 ionic strength on degree of separation

(4) 50mM Lin acid hydrogen Er Na ︰ acetonitrile=15 ︰ 85, add triethylamine 0.01%.Fig. 4 a intermediate 2 is separated with impurity peaks better, and main peak theoretical cam curve reaches 50000; Fig. 4 b product 1 can be better separated with each impurity, and theoretical cam curve is greater than 15000.

2, the selection of wavelength

Prepare respectively certain density AV45 and intermediate acetonitrile solution, with acetonitrile, make blank, from 200mm ~ 500mm scanning, scanning spectra is shown in Fig. 5.Both uv absorption are large and peak shape is mild near 325mm, therefore select 325 mm as detecting wavelength.

3, the selection of solvent and stability test

Take a certain amount of AV45, with acetonitrile, ethanol, the dissolution with solvents that methyl alcohol etc. are different.AV45 is less stable in ethanol, methyl alcohol, and room temperature is placed after 3d, degrades, and impurity peaks increases, and main peak content reduces (Fig. 6 b, 6c); Relatively stable in acetonitrile solution, room temperature is placed after 3d AV45 content and is still greater than 95% (Fig. 6 a).Therefore select acetonitrile to make solvent, analyze and completed on the same day.

4, specificity test

Precision takes 3 parts of AV45 reference substances, adds respectively 3 kinds of solution: (1) 6 mol/L NaOH solution 5 mL; (2) 6 mol/L HCl solution 5 mL; (3) 30% H ₂o ₂5 mL.Above-mentioned solution is placed in to boiling water bath and adds respectively heat damage 1h.Adjust respectively pH to neutral, evaporated under reduced pressure, dissolves with acetonitrile, is diluted to the sample liquid of 0.6mg/mL, filters again.By above-mentioned chromatographic condition sample introduction.As a result, each catabolite all can reach completely separated with major component peak.

5, linear relationship and detectability test ?

Accurately take a certain amount of reference substance AV45 appropriate, be made into 1.0mg/mL acetonitrile solution and make storing solution; Precision measures 0.2,0.4,0.6,0.8,1.0mL storing solution, uses dilution in acetonitrile constant volume, is made into the AV45 acetonitrile solution of 200,400,600,800,1000 μ g/mL.Sample introduction 10 μ L, map to sample concentration with integral area respectively.Gained regression equation is y=3.2E-8X-0.0087, R ²=0.9992.In the scope of 200 ~ 1000 μ g/mL, present good linear relationship.AV45 minimum detectable activity is 0.1ng (S/N >=3).

6, precision test

Under definite chromatographic condition respectively at 1d in and continuously in 3d sample introduction measure, RSD in a few days and is in the daytime respectively 0.49% and 1.2% (n=6).

7, the mensuration of sample size

The reference substance AV45 that gets dry constant weight is appropriate, accurately weighed, with acetonitrile, is mixed with the solution containing AV45 0.6mg/mL, and solution, counts W contrast in contrast; Another sample thief is appropriate, with acetonitrile, be made into containing the tested AV45 sample solution that is 0.6mg/mL as need testing solution, count W sample, by above-mentioned chromatographic condition, getting respectively 10 μ L injecting chromatographs measures, measure gained peak area and be respectively S contrast, S sample, by external standard method, calculate AV45 content: (S sample/S contrast) * (W contrast/W sample) * 100%.

Sample determination the results are shown in Table 2, and chromatogram is shown in Fig. 7.

8, its related substances is measured

It is appropriate that precision takes AV45, with acetonitrile dissolve be prepared into every 1 mL containing the solution of 5.0 mg as need testing solution; Precision measures 1 mL need testing solution and is placed in 100 mL volumetric flasks, adds dilution in acetonitrile to scale, shakes up, in contrast liquid.Get contrast liquid 10 μ L injecting chromatographs, regulate instrumental sensitivity, make major component peak height be about 20%～25% of registering instrument full scale; Measure test liquid 10 μ L, injecting chromatograph, records chromatogram to 4 times of main peak retention time, gets the peak area of main peak and related substance again, calculates the content of related substance by Self-control method, the results are shown in Table 2.

Table 2 sample size and its related substances are measured

Claims

1. a content analysis method of A beta plaque imaging agent precursor AV45, is characterized in that chromatographic condition:

Instrument: WATERS 600 type high performance liquid chromatographs, 2487 UV-detector, chromatographic column Inertsil C18 post, 5 μ m, 4.6 * 250mm;

Mobile phase: 50mM Lin acid hydrogen Er Na ︰ acetonitrile volume ratio=15 ︰ 85, adds triethylamine v/v 0.01%, also degassed through the organic filter membrane suction filtration of 0.45 μ m before using;

Flow velocity: 0.8mL/min;

The sampling volume of AV45 sample: 10 μ L; Column temperature: room temperature; Detect wavelength: 325nm.