A method for detecting the radiochemical purity of molecular probe [99mTc]Tc-BAT-P-DTBZ

Abstract

The present invention provides a method for detecting the radiochemical purity of a molecular probe [99mTc]Tc-BAT-P-DTBZ, comprising the following steps: 99m Tc]Tc-BAT-P-DTBZ solution is the test solution; according to the thin layer chromatography method, the chromatographic conditions are: the stationary phase is normal phase chromatography paper, the developing solvent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:0.5-1.0; the above method can achieve the purpose of simple, rapid and low-cost detection, and the measured radiochemical purity is highly consistent with the HPLC determination result. In addition, it can also achieve [ 99m Tc]Tc-BAT-P-DTBZ and the small amount of radioactive impurities that may exist during its preparation process are effectively separated.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting the radiochemical purity of a molecular probe [99mTc]Tc-BAT-P-DTBZ. Background Art

[0002] The type II vesicular monoamine transporter (VMAT2) is a membrane protein located on the surface of vesicles in presynaptic neurons. Its function is to transport monoamine neurotransmitters synthesized by neurons into vesicles for release and utilization, thereby regulating the concentration of monoamine neurotransmitters in neurons. Molecular biological studies have shown that VMAT2 is mainly distributed in the central nervous system and pancreatic tissue of humans and mammals. Changes in VMAT2 are associated with a variety of diseases, such as diabetes and Parkinson's disease (PD). Tracing imaging studies of VMAT2 using radioactive molecular probes can provide valuable information for related diseases. Recent studies have shown that radioactive molecular probes [technetium 99m Tc]-diaminodimercapto-propyl-dihydrotetrabenazine ([ 99m Tc]Tc-BAT-P-DTBZ) has good in vitro stability, lipophilicity and biological activity, has good affinity for VMAT2, and has the potential to be used for tracing imaging of VMAT2 (Arabian Journal of Chemistry, 2023, 16, 4: 104572). Radiochemical purity is one of the important quality indicators of radioactive molecular probes. Molecular probes with higher radiochemical purity have better tracing effects. Currently, the reported radioactive molecular probes [ 99m The radiochemical purity of Tc]Tc-BAT-P-DTBZ was determined by high performance liquid chromatography (HPLC), using a Waters Symmetry C18 column, mobile phase: acetonitrile / water / triethylamine = 48:52:0.1 (V / V / V), detector: Radiomatic 610TR radioactivity detector, flow rate: 1 mL / min, run time: 20 min (Arabian Journal of Chemistry, 2023, 16, 4: 104572). This method has the disadvantages of relatively complex experimental pretreatment, high cost, and long time consumption, which is not conducive to molecular probes [ 99m Therefore, a new simple, fast and low-cost [ 99m The radiochemical purity detection method of Tc]Tc-BAT-P-DTBZ is of great significance. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide a new simple, fast and low-cost [99m Tc]Tc-BAT-P-DTBZ radiochemical purity detection method.

[0004] To this end, the present invention provides the following technical solutions:

[0005] A molecular probe 99m The radiochemical purity detection method of [Tc]Tc-BAT-P-DTBZ comprises the following steps:

[0006] To contain [ 99m Tc]Tc-BAT-P-DTBZ solution was the test solution;

[0007] According to the thin layer chromatography method, the chromatographic conditions are as follows: the stationary phase is normal phase chromatography paper, the developing solvent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:0.5-1.0.

[0008] Optionally, the volume ratio of dichloromethane to methanol is 10:0.8.

[0009] Optionally, the normal phase chromatography paper is a polyamide film.

[0010] Optionally, the test solution is spotted on the stationary phase, and after drying, one end of the stationary phase with the spotted sample is placed in the developing agent, keeping the spotted point above the developing agent liquid surface. After the developing agent is developed, the stationary phase is removed, dried, the radioactivity count is obtained, and the radiochemical purity is calculated.

[0011] Optionally, the test solution is spotted at a distance of 1-1.5 cm from one end of the stationary phase.

[0012] Optionally, the test solution is spotted at a position 1 cm away from one end of the stationary phase.

[0013] Optionally, after 5-15 minutes of development, the stationary phase is removed.

[0014] Optionally, after developing for 5 minutes, the stationary phase is removed.

[0015] Optionally, the length of the stationary phase is 10-15 cm.

[0016] The technical solution of the present invention has the following advantages:

[0017] 1. A molecular probe provided by the present invention [ 99m The radiochemical purity detection method of Tc]Tc-BAT-P-DTBZ comprises the following steps: 99mTc]Tc-BAT-P-DTBZ solution is the test solution; according to the thin layer chromatography method, the chromatographic conditions are: the stationary phase is normal phase chromatography paper, the developing solvent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:0.5-1.0; the above method can achieve the purpose of simple, rapid and low-cost detection, and the measured radiochemical purity is highly consistent with the result of the existing HPLC method, and the method of the present invention can also achieve [ 99m Tc]Tc-BAT-P-DTBZ and the small amount of radioactive impurities that may exist in its preparation process [ 99m Tc]-glucoheptonic acid sodium ([ 99m Tc]Tc-GH), high technetium [ 99m Sodium Tc] 99m TcO4), technetium [ 99m Tc]-colloid ([ 99m Effective separation between Tc]Tc-radiocolloid). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The TLC method of the present invention and the HPLC method in the experimental example of the present invention are used to determine [ 99m Comparison of the correlation between radiochemical purity of Tc]Tc-BAT-P-DTBZ (with Na 99m TcO4 as a radioactive impurity);

[0020] Figure 2 The TLC method of the present invention and the HPLC method in the experimental example of the present invention are used to determine [ 99m Comparison of the correlation between radiochemical purity of Tc]Tc-BAT-P-DTBZ (with [ 99m Tc]Tc-GH as a radioactive impurity);

[0021] Figure 3 The TLC method of the present invention and the HPLC method in the experimental example of the present invention are used to determine [ 99m Comparison of the correlation between radiochemical purity of Tc]Tc-BAT-P-DTBZ (with [ 99m Tc]Tc-radiocolloid as a radioactive impurity). DETAILED DESCRIPTION

[0022] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0023] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0024] In the following examples, (1) [ 99m The preparation method of the Tc]Tc-BAT-P-DTBZ test solution was as follows: 2 mL of a mixed aqueous solution containing trisodium citrate (30 mg), citric acid (0.5 mg), GH (10 mg), EDTA-2Na (1 mg), SnCl2·2H2O (20 μg) and the labeled precursor 9-BAT-P-DTBZ (50 μg) was prepared, and then the freshly washed Na 99m TcO4 solution (37Mbq) was mixed and allowed to stand for 5 minutes, then heated in a boiling water bath for 30 minutes and cooled to complete the labeling reaction. The preparation of the labeling precursor 9-BAT-P-DTBZ can be found in Chinese patent document CN115536705;

[0025] (2)[ 99m Tc]Tc-GH was prepared by preparing 2 mL of a mixed aqueous solution containing trisodium citrate (30 mg), citric acid (0.5 mg), GH (10 mg), EDTA-2Na (1 mg), and SnCl2·2H2O (20 μg), and then adding freshly washed Na 99m TcO4 solution (37Mbq) was mixed and allowed to stand for 5 minutes, then heated in a boiling water bath for 30 minutes and cooled to complete the labeling reaction;

[0026] (3)[ 99m Tc]Tc-radiocolloid was prepared by: freshly washed Na 99m TcO4 solution (37Mbq) was added with an appropriate amount of SnCl2·2H2O (20μg) and mixed thoroughly to obtain the product;

[0027] (4) TLC determination 99m The radiochemical purity (RCP) of Tc]Tc-BAT-P-DTBZ was calculated as follows: RCP = (radioactivity counts in the ninth section of the chromatographic strip + radioactivity counts in the tenth section of the chromatographic strip) / total radioactivity counts in the tenth section of the chromatographic strip × 100%; fValue = a / b; a is the distance from the center of the spot to the origin, and b is the distance from the solvent front to the origin.

[0028] (5) HPLC determination 99m The radiochemical purity (RCP) of Tc-BAT-P-DTBZ was calculated as follows: RCP = [ 99m Tc]Tc-BAT-P-DTBZ radioactive peak area / ([ 99m Tc]Tc-BAT-P-DTBZ radioactive peak area + radioactive impurity peak area) × 100%.

[0029] The solvents such as dichloromethane and methanol used to prepare the developing agents in the following examples were of analytical grade.

[0030] Example 1

[0031] This embodiment provides a molecular probe [ 99m The radiochemical purity detection method of [Tc]Tc-BAT-P-DTBZ comprises the following steps:

[0032] To contain [ 99m Tc]Tc-BAT-P-DTBZ solution is the test solution (the test solution prepared in (1) above);

[0033] According to the thin layer chromatography (TLC) method, the chromatographic conditions are as follows: the stationary phase is a polyamide thin film chromatography strip (length 11 cm, width about 1 cm), the developing solvent is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:0.8;

[0034] Take a polyamide film chromatography strip (11cm×1cm) and use a pencil to lightly draw a mark line 1cm away from the bottom on one side, mark the center of the mark line as the sample spot mark point, and then take the test solution and spot it at the sample spot mark point; after the sample is dried, the chromatography strip is developed in a glass test tube with dichloromethane-methanol (10:0.8, V / V) as the developing agent, keeping the sample spot above the developing agent liquid surface. After about 5 minutes, take out the chromatography strip, dry it, cut the stationary phase chromatography paper strip into ten sections of approximately the same length and place them in radioimmunoassay tubes respectively, measure the radioactivity count with a γ counter, and analyze the peak shift value R f , calculate the radiochemical purity. The radioactivity counting results are shown in the following table:

[0035] Table 1

[0036]

[0037] The test results are: radioactive impurity Na 99m TcO4, [ 99m Tc]Tc-GH, [99m R of Tc]Tc-radiocolloid f The values ​​are all 0.1-0.2, [ 99m R of Tc]Tc-BAT-P-DTBZ f The value is 0.9-1.0, [ 99m The separation between Tc]Tc-BAT-P-DTBZ and impurities was good and there was no tailing phenomenon.

[0038] Example 2

[0039] The main difference between this example and Example 1 is that the volume ratio of dichloromethane to methanol is 10:0.5. A polyamide film chromatography strip (11 cm x 1 cm) was taken and a line was lightly drawn on one side with a pencil, 1.5 cm from the bottom. After approximately 15 minutes, the strip was removed. The length of the stationary phase was 15 cm. The radioactivity count results are shown in the table below:

[0040] Table 2

[0041]

[0042] The test results are: radioactive impurity Na 99m TcO4, [ 99m Tc]Tc-GH, [ 99m R of Tc]Tc-radiocolloid f The values ​​are all 0.1-0.2, [ 99m R of Tc]Tc-BAT-P-DTBZ f The value is 0.8-1.0, [ 99m The separation degree between Tc]Tc-BAT-P-DTBZ and impurities was good and the tailing phenomenon was not obvious.

[0043] Example 3

[0044] The main difference between this example and Example 1 is that the volume ratio of dichloromethane to methanol is 10:1.0. The length of the stationary phase is 10 cm. The radioactivity counting results are shown in the following table:

[0045] Table 3

[0046]

[0047] The test results are: radioactive impurity Na 99m TcO4, [ 99m Tc]Tc-GH, [ 99m R of Tc]Tc-radiocolloid f The values ​​are all 0.1-0.2, [ 99m R of Tc]Tc-BAT-P-DTBZf The value is 0.9-1.0, [ 99m The separation between Tc]Tc-BAT-P-DTBZ and impurities was good and there was no tailing phenomenon.

[0048] Comparative Example 1

[0049] The difference between this comparative example and Example 1 is that the developing solvent is toluene-acetonitrile-triethylamine (50:50:0.5, V / V / V). The radioactivity counting results are shown in the following table:

[0050] Table 4

[0051]

[0052] The test results are: radioactive impurity Na 99m TcO4, [ 99m Tc]Tc-GH, [ 99m R of Tc]Tc-radiocolloid f The values ​​are all between 0.1 and 0.7. 99m R of Tc]Tc-BAT-P-DTBZ f The value is 0.5-1.0, [ 99m The separation degree between Tc]Tc-BAT-P-DTBZ and impurities was poor and the tailing phenomenon was obvious.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 1 is that the developing solvent is toluene-acetonitrile-triethylamine (75:25:0.5, V / V / V). The radioactivity counting results are shown in the following table:

[0055] Table 5

[0056]

[0057] The test results are: radioactive impurity Na 99m TcO4, [ 99m Tc]Tc-GH, [ 99m R of Tc]Tc-radiocolloid f The values ​​are all 0.1-0.2, [ 99m R of Tc]Tc-BAT-P-DTBZ f The value is 0.7-1.0, [ 99m The separation degree between Tc]Tc-BAT-P-DTBZ and impurities is good, but the tailing phenomenon is obvious.

[0058] Comparative Example 3

[0059] The difference between this comparative example and Example 1 is that the developing solvent is ethyl acetate-methanol (10:0.8, V / V). The radioactivity counting results are shown in the following table:

[0060] Table 6

[0061]

[0062] The test results are: radioactive impurity Na 99m TcO4, [ 99m Tc]Tc-GH, [ 99m R of Tc]Tc-radiocolloid f The values ​​are all 0.1-0.2, [ 99m R of Tc]Tc-BAT-P-DTBZ f The value is 0.6-1.0, [ 99m The separation degree between Tc]Tc-BAT-P-DTBZ and impurities is good, but the tailing phenomenon is obvious.

[0063] Experimental example

[0064] The method of the present invention is similar to the HPLC method for determining [ 99m Comparison of the correlation between radiochemical purity of Tc]Tc-BAT-P-DTBZ

[0065] (1) Take Na 99m TcO4 as a radioactive impurity

[0066] Preparation of test solution: in high purity [ 99m A certain proportion of Na was added to the Tc-BAT-P-DTBZ solution. 99m TcO4 solution was used as a radioactive impurity to obtain various radiochemical purities [ 99m Tc]Tc-BAT-P-DTBZ solution.

[0067] TLC determination of radiochemical purity: The test solution was subjected to the thin layer chromatography conditions in Example 1, and the radiochemical purity was calculated.

[0068] HPLC determination of radiochemical purity: 20 μL of the sample solution was analyzed on an analytical HPLC system equipped with a radioactivity detector. Chromatographic conditions were: a Waters Symmetry C18 reversed-phase column (4.6×150 mm, 5 μm); mobile phases consisting of phase A (acetonitrile containing 0.1 v / v% triethanolamine (TEA)) and phase B (water containing 0.1 v / v% TEA), with an A:B ratio of 48:52 (v / v), and isocratic elution; flow rate 1.0 mL / min. Radiochemical purity was calculated based on the peak area ratios in the resulting chromatographic spectrum.

[0069] The correlation between the TLC and HPLC results of the present invention was established by taking the radiochemical purity measured by TLC of the present invention as the abscissa and the radiochemical purity measured by HPLC as the ordinate. The results are as follows: Figure 1 As shown in the figure, the results of the two methods were highly consistent, with a correlation coefficient of 0.999.

[0070] (2) 99m Tc]Tc-GH as a radioactive impurity

[0071] Preparation of test solution: in high purity [ 99m Tc]Tc-BAT-P-DTBZ solution was added with a certain proportion of [ 99m Tc]Tc-GH solution as a radioactive impurity, various radiochemical purities of [ 99m Tc]Tc-BAT-P-DTBZ solution.

[0072] TLC determination of radiochemical purity: The test solution was tested using the optimal thin layer chromatography conditions in Example 1, and then the radiochemical purity was calculated.

[0073] HPLC determination of radiochemical purity: 20 μL of the sample solution was analyzed on an analytical HPLC system equipped with a radioactivity detector. Chromatographic conditions were: a Waters Symmetry C18 reversed-phase column (4.6 × 150 mm, 5 μm); mobile phases consisting of phase A (acetonitrile containing 0.1% TEA) and phase B (water containing 0.1% TEA), with an A:B ratio of 48:52 (v / v), and isocratic elution; flow rate 1.0 mL / min. Radiochemical purity was calculated based on the peak area ratios in the resulting chromatographic spectrum.

[0074] The correlation between TLC and HPLC results was established by taking the radiochemical purity measured by TLC of the present invention as the abscissa and the radiochemical purity measured by HPLC as the ordinate. The results were as follows: Figure 2 As shown in the figure, the results of the two methods were highly consistent, with a correlation coefficient of 0.999.

[0075] (3) 99m Tc]Tc-radiocolloid as a radioactive impurity

[0076] Preparation of test solution: in high purity [ 99m Tc]Tc-BAT-P-DTBZ solution was added with a certain proportion of [ 99m Tc]Tc-radiocolloid solution as a radioactive impurity to obtain various radiochemical purities of [ 99m Tc]Tc-BAT-P-DTBZ solution.

[0077] TLC determination of radiochemical purity: The test solution was tested using the optimal thin layer chromatography conditions in Example 1, and then the radiochemical purity was calculated.

[0078] HPLC determination of radiochemical purity: 20 μL of the sample solution was analyzed on an analytical HPLC system equipped with a radioactivity detector. Chromatographic conditions were: a Waters Symmetry C18 reversed-phase column (4.6 × 150 mm, 5 μm); mobile phases consisting of phase A (acetonitrile containing 0.1% TEA) and phase B (water containing 0.1% TEA), with an A:B ratio of 48:52 (v / v), and isocratic elution; flow rate 1.0 mL / min. Radiochemical purity was calculated based on the peak area ratios in the resulting chromatographic spectrum.

[0079] The correlation between TLC and HPLC results was established by taking the radiochemical purity measured by TLC of the present invention as the abscissa and the radiochemical purity measured by HPLC as the ordinate. The results were as follows: Figure 3 As shown in the figure, the results of the two methods were highly consistent, with a correlation coefficient of 0.9987.

[0080] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A molecular probe 99m The radiochemical purity detection method of Tc-BAT-P-DTBZ is characterized by: The steps include: To contain 99m The solution of Tc-BAT-P-DTBZ is the test solution; According to the thin layer chromatography method, the chromatographic conditions are as follows: the stationary phase is normal phase chromatography paper, the developing solvent is a mixture of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 10:0.5-1.0; the normal phase chromatography paper is a polyamide film; The molecular probe 99m Tc-BAT-P-DTBZ is a molecular probe [ 99m Tc]-diaminodimercapto-propyl-dihydrotetrabenazine.

2. The molecular probe according to claim 1 99m The radiochemical purity detection method of Tc-BAT-P-DTBZ is characterized by: The volume ratio of dichloromethane to methanol is 10:0.

8.

3. The molecular probe according to claim 1 99m The radiochemical purity detection method of Tc-BAT-P-DTBZ is characterized by: The sample solution is spotted on the stationary phase. After drying, one end of the stationary phase where the sample is spotted is placed in the developing agent, keeping the spotted point above the developing agent liquid surface. After the developing agent is developed, the stationary phase is removed, dried, radioactivity counts are obtained, and the radiochemical purity is calculated.

4. The molecular probe according to claim 3 99m The radiochemical purity detection method of Tc-BAT-P-DTBZ is characterized by: The test solution was spotted at a distance of 1-1.5 cm from one end of the stationary phase.

5. The molecular probe according to claim 4 99m The radiochemical purity detection method of Tc-BAT-P-DTBZ is characterized by: The test solution was spotted at a position 1 cm away from one end of the stationary phase.

6. The molecular probe according to any one of claims 4 to 5 99m The radiochemical purity detection method of Tc-BAT-P-DTBZ is characterized by: After 5-15 min of development, the stationary phase was removed.

7. The molecular probe according to claim 6 99m The radiochemical purity detection method of Tc-BAT-P-DTBZ is characterized by: After 5 min of development, the stationary phase was removed.

8. The molecular probe according to any one of claims 4 to 5. 99m The radiochemical purity detection method of Tc-BAT-P-DTBZ is characterized by: The length of the stationary phase is 10-15 cm.

Images