A method for detecting the radiochemical purity of an actinium [ 225 Ac] conjugate

Abstract

The application belongs to the field of analytical chemistry detection, and particularly relates to a method for detecting radiochemical purity of an actinium 225 Ac] conjugate. The method adopts high performance liquid chromatography combined with a gamma counter, a mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a mixed solution of buffer salt and cationic pair reagent, and the mobile phase B is acetonitrile. 225 The detection method can accurately detect the radiochemical purity of the actinium 225 Ac] conjugate, is stable and reliable, has high sensitivity, and is of great significance for quality control and research of the actinium [Ac] conjugate product.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry detection, specifically relating to an actinide [ 225 Methods for determining the radiochemical purity of Ac] conjugates. Background Technology

[0002] Radioactive medical isotope drugs are a special class of drugs containing radioactive isotopes for medical diagnosis and treatment, enabling early screening, diagnosis, precision treatment, and pain relief for malignant tumors. 225 Actinium is a 100% alpha decay radionuclide with a half-life of t. 1 / 2 It takes 9.96 days to decay into the stable nuclide Bi-209 (nearly stable), undergoing 4 α decays and 2 β decays, releasing α particles with a total energy of 27.5 MeV. This results in a large radiation dose from the parent nuclide to the target site and significant biological effects. It is considered the most suitable α-targeted therapeutic nuclide for tumor diagnosis and treatment, especially for patients who have failed other treatments, particularly those with advanced multi-metastatic tumors.

[0003] The Chinese Pharmacopoeia, the United States Pharmacopoeia, and the European Pharmacopoeia have not yet addressed this issue. 225 Quality standards for actinium radiopharmaceuticals. The IAEA (International Atomic Energy Agency) recommends using HPLC for detection. 225 The radiochemical purity of actinium radiopharmaceuticals should be ≥95%. Radiochemical purity refers to the percentage (%) of the radioactivity of a radionuclide in a specific chemical form relative to the total radioactivity of that radionuclide.

[0004] Currently, in relevant literature both domestically and internationally 225 Compounds labeled with Ac radionuclides were all tested for radiochemical purity using a method with 0.1% trifluoroacetic acid in water and acetonitrile as the mobile phase. For details, please refer to the IAEA (Production and Quality Control of Actinium-225 Radiopharmaceuticals). Under these mobile phase conditions, if the labeled compound is strongly acidic, it will lead to… 225 Actinium ion delabeling leads to a serious underestimation of its radiochemical purity, thus affecting the judgment of labeling results. Summary of the Invention

[0005] On the one hand, the present invention provides an actinide [ 225 A method for the radiochemical purity detection of Ac] conjugates, wherein the method employs high performance liquid chromatography, and the mobile phase comprises mobile phase A and mobile phase B, and gradient elution is performed;

[0006] The mobile phase A mainly consists of a mixed solution of buffer salt and cation-pair reagent;

[0007] The mobile phase B comprises one or more of acetonitrile, methanol, and tetrahydrofuran;

[0008] The high-performance liquid chromatography column is an octadecyl bonded silica column;

[0009] The 225 Ac conjugates are selected from compounds 1, 2, 3 and 4.

[0010] The chemical formula of compound 1 is shown in Formula I:

[0011] Formula I;

[0012] The chemical formula of compound 2 is shown in Formula II:

[0013] Formula II;

[0014] The chemical formula of compound 3 is shown in Formula III:

[0015] Formula III;

[0016] The chemical formula of compound 4 is shown in Formula IV:

[0017] Formula IV.

[0018] In some embodiments, the buffer salt in the mobile phase A is selected from acetate; and / or the cation-pairing reagent is selected from tetrabutylammonium salt.

[0019] In some embodiments, the acetate includes sodium acetate and ammonium acetate.

[0020] In some embodiments, the tetrabutylammonium salt includes tetraethylammonium chloride, tetrabutylammonium bisulfate, tetrabutylammonium bromide, and tetrabutylammonium hydroxide.

[0021] In some embodiments, the concentration of the cation-pairing reagent in the mobile phase A is 2-8 mmol / L; in some embodiments, the concentration of the buffer salt is 20-30 mmol / L.

[0022] In some embodiments, the pH of the mobile phase A is 5.5-6.5.

[0023] In some embodiments, the chromatographic column of the high-performance liquid chromatography is selected from Atlantis T3, Luna C18, Ultimate C18 and Premier Peptide CSH C18; in some embodiments, the chromatographic column of the high-performance liquid chromatography is Atlantis T3.

[0024] In some embodiments, the elution process of the mobile phase, with the total volume of the mobile phase being 100%, includes:

[0025] From 0 to 2 minutes, the volume fraction of mobile phase A is 90% to 100%, and the volume fraction of mobile phase B is 0% to 10%.

[0026] Between 2 and 17 minutes, the volume fraction of mobile phase A decreased from 90% to 100% to 30% to 40%, while the volume fraction of mobile phase B increased from 0% to 10% to 50% to 70%.

[0027] In some embodiments, the elution process of the mobile phase, with the total volume of the mobile phase being 100%, includes:

[0028] From 0 to 2 minutes, the volume fraction of mobile phase A was 95%, and the volume fraction of mobile phase B was 5%.

[0029] Between 2 and 17 minutes, the volume fraction of mobile phase A decreased from 95% to 35%, while the volume fraction of mobile phase B increased from 5% to 65%.

[0030] In some implementations, the detector of the detection method is a gamma counter.

[0031] In some implementations, the fraction collector of the method collects fractions at a frequency of 55-65 s / sample, preferably 60 s / sample.

[0032] In some implementations, the injection activity of the analyte is 3.7~56.8 kBq, preferably 37 kBq.

[0033] In some implementations, the 225 The equilibrium time of Ac is 221 The equilibration time is 45 min-24 h, preferably 15 h, according to the Fr meter.

[0034] In some embodiments, the flow rate of the mobile phase is 0.8-1.2 ml / min, preferably 1.0 ml / min.

[0035] In some implementations, the column temperature is 25-35°C, preferably 30°C.

[0036] In some implementations, the 225 The method for calculating the radiochemical purity of Ac conjugates is: Radiochemical purity = 100% - 225 Actinium-DTPA (%) - Other radioactive unknown impurities (%); The percentage in the formula is calculated as: radioactivity count rate of each fraction / radioactivity count rate of the total fraction (CPM) × 100%; fractions with a content of less than 0.1% are ignored.

[0037] On the one hand, the present invention provides the detection method described above in the presence of 225 Applications of Ac conjugates in quality testing or quality control of products.

[0038] In some implementation schemes, containing 225 The composition of Ac conjugate products includes: 225 Ac conjugate, not 225 Ac-labeled conjugate precursor, DTPA, vitamin C and / or sodium vitamin C, gentic acid and / or sodium gentinate, sodium acetate.

[0039] The beneficial effects of this invention include:

[0040] The detection method of the present invention is capable of... 225 Ac conjugates are effectively separated from radioactive impurities, with sharp peaks and good separation from the main peak. This allows for accurate detection. 225 Ac conjugates exhibit high radiochemical purity, stability, reliability, high sensitivity, and a good linear range; and are effective against a variety of... 225 The detection method for the radiochemical purity of Ac conjugates is applicable, and the measured radiochemical purity is consistent with the actual results. The detection method of this invention comprises actinide […]. 225 This provides a basis for the quality control and research of Ac] conjugate products. Attached Figure Description

[0041] Figure 1 The liquid phase-fraction-counter plot for the test solution of compound 1, using 0.1% trifluoroacetic acid and acetonitrile as the mobile phase as recommended by the IAEA in Example 1, is shown.

[0042] Figure 2 The liquid phase-fraction-counter diagram for detecting water using the method of the present invention in Example 1 is shown.

[0043] Figure 3 This is a liquid phase-fraction-counter plot of the test solution of compound 1 detected by the method of the present invention in Example 1.

[0044] Figure 4 In Example 1, the method of the present invention detects typical impurities. 225 Liquid phase-fraction-counter plot of actinium-DTPA impurity control solution.

[0045] Figure 5 In Example 1, the method of the present invention is used to detect the spiked sample solution of compound 1. 225 Liquid phase-fraction-counter plot of actinium-DTPA mixed solution.

[0046] Figure 6 for 225A schematic diagram of the decay chain of Ac.

[0047] Figure 7 The equation is a linear regression equation for the radioactivity of compound 1 and the count rate (CPM). Detailed Implementation

[0048] The above embodiments are merely preferred embodiments of this application and should not be construed as limiting the scope of protection of this application. Any non-substantial changes and substitutions made by those skilled in the art based on this application shall fall within the scope of protection claimed by this application.

[0049] As used herein, all features or conditions defined by numerical ranges or percentage ranges mentioned by endpoints are for the sake of brevity and convenience only. Therefore, descriptions of numerical ranges or percentage ranges should be considered to cover and specifically disclose all possible subranges and all individual integer and fractional values ​​within those ranges, particularly integer values. For example, a description of a range “1 to 8” should be considered to specifically disclose all subranges such as 1 to 7, 2 to 8, 3 to 6, 3 to 6, 4 to 8, 3 to 8… etc., particularly subranges defined by all integer values, and should be considered to specifically disclose individual values ​​within those ranges such as 1, 2, 3, 4, 5, 6, 7, 8, etc., regardless of whether these ranges or individual values ​​are explicitly stated. Similarly, a description of a range “between 1 and 8” should also be considered to specifically disclose all ranges such as 1 to 8, 1 to 7, 2 to 8, 3 to 6, 3 to 6, 4 to 8, 3 to 8, etc., and include the endpoint values ​​of these ranges, such as individual values ​​1, 2, 3, 4, 5, 6, 7, 8, etc. Furthermore, when a parameter is described as an integer greater than or equal to 2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0050] As used herein, the term “about” refers to an approximation, within approximately or near a range. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the limits above or below the provided numerical value. Generally, the term “about” is used herein to mean a numerical value that varies by 10% above or below the provided value. On the other hand, the term “about” refers to adding or subtracting 20% ​​of the value of the number it modifies. For example, “about 50%” means within the range of 45%–55%. It should also be understood that all integers and fractions are considered to be modified by the term “about.” In this document, numerical values ​​are to be understood to have the precision of the stated numerical value in significant digits, to the extent that the inventive purpose may be achieved. For example, the number 40.0 can be understood to cover a range from 39.50 to 40.49.

[0051] As used herein, the terms “comprising,” “including,” or “containing” are non-exclusive or open-ended terms intended to indicate that a combination (e.g., an apparatus, composition, method, etc.) includes the listed elements (e.g., units of an apparatus, components of a composition, substantial steps of a method, etc.) but does not exclude other elements.

[0052] As used herein, the term "and / or" ("and / or") means and covers any and all possible combinations of one or more associated listed items. When used in a list of two or more items, the term "and / or" ("and / or") indicates that any one of the listed items may be included alone, or may include any combination of two or more listed items. For example, if a group, combination, or composition is described as including components A, B, and / or C, then the composition may include A alone, include B alone, include C alone, include a combination of A and B, include a combination of A and C, include a combination of B and C, or include a combination of A, B, and C.

[0053] The sources of the raw materials used in the following specific embodiments of the present invention are shown in Table 1; the other raw materials, reagents, solvents, etc. can all be obtained commercially.

[0054] Table 1. Sources of raw materials used in the examples

[0055]

[0056]

[0057] #In the “Specifications / Content” column of the table, the “Specifications / Content” for the precursors of compounds 1-4 and DTPA refers to the mass content; the “Specifications / Content” for compound 1 and 225 Actinide-DTPA refers to the radiochemical purity.

[0058] ^In the table, the precursor of compound 1 was prepared in-house. The preparation method included: coupling of amino acids according to the standard Fmoc solid-phase synthesis method, and finally coupling with HBTU-activated DOTA tritert-butyl ester. (Alternatively, the detailed preparation process of DXJ137 in patent publication number CN112062695A can be referred to).

[0059] Example 1

[0060] The solution containing compound 1 was used as the test solution.

[0061] Preparation of the test solution:

[0062] Preparation of radionuclide solution: Take nitric acid 225 An appropriate amount of actinium nuclide was dissolved and diluted to 37 kBq / μl with 0.1 mol / L hydrochloric acid solution.

[0063] Preparation of the precursor solution of compound 1: Take an appropriate amount of the precursor of compound 1, dissolve it in water and dilute it to 1 nmol / μl.

[0064] Buffer solution preparation: Take an appropriate amount of sodium acetate, dissolve it in aqueous solution until the sodium acetate concentration is 1 mol / L, and adjust the pH to 7.0 with 1 mol / L sodium hydroxide.

[0065] DTPA solution preparation: Take an appropriate amount of DTPA, dissolve it in water by sonication and dilute it to 0.1 mg / ml.

[0066] Diluent stock solution: Take appropriate amounts of vitamin C and gentian acid, dissolve and dilute with water to an aqueous solution containing 281.6 mg of vitamin C and 1 mg of gentian acid per 1 ml of solution, and adjust the pH to 5.8 with 1 mol / L sodium hydroxide solution.

[0067] Diluent: Take an appropriate amount of DTPA, dissolve it by sonication with the stock solution of diluent, and dilute it to an aqueous solution containing 281.6 mg of vitamin C, 1 mg of gentian acid, and 0.2 mg of DTPA per 1 ml.

[0068] Test solution: Take 30 μl of the radionuclide solution, 360 μl of the precursor solution of compound 1, and 360 μl of the buffer solution, react at 95℃ for 15 min, add 420 μl of diluent, and shake well to obtain the test solution.

[0069] 225 Actinium-DTPA impurity control solution: Take 30 μl of the radionuclide solution and 360 μl of the DTPA solution, react at 95℃ for 15 min, add 420 μl of diluent, and shake well to obtain the solution. 225 Actinium-DTPA impurity control solution.

[0070] High performance liquid chromatography, chromatographic conditions as follows:

[0071] The chromatographic column was an octadecyl bonded silica column, Atlantis T3 (4.6×150mm, 3μm).

[0072] Mobile phase A is an aqueous solution containing 5 mmol / L tetrabutylammonium hydrogen sulfate and 25 mmol / L sodium acetate, with the pH adjusted to 6.0 using glacial acetic acid; mobile phase B is acetonitrile.

[0073] The mobile phase was eluted using the following program: 0–2 min, mobile phase A was 95% by volume and mobile phase B was 5% by volume; 2–17 min, mobile phase A decreased from 95% to 35% by volume and mobile phase B increased from 5% to 65% by volume.

[0074] The fraction collector sampling frequency was 60 s / sample; the mobile phase flow rate was 1.0 ml / min; and the column temperature was 30℃.

[0075] Take the test solution, with an injection activity of approximately 37 kBq; after collecting all fractions, let it stand for 15 h, and then use a gamma counter (Revvity 2470) to detect the activity in each fraction. 221 Radioactivity count rate of francium.

[0076] A curve was plotted with the radioactivity count rate of each fraction (CPM) / total fraction radioactivity count rate (CPM) × 100% as the ordinate and the order of each fraction as the abscissa, and the radiochemical purity was calculated.

[0077] Radiochemical purity = 100% 225 Actinium-DTPA (%) - Other radioactive unknown impurities (%), ignoring fractions with a content of less than 0.1%.

[0078] The results of the radioactivity count rate and the proportion of radioactivity count rate to total radioactivity are shown in Table 2:

[0079] Table 2 Results of radioactivity count rate and the proportion of radioactivity count rate to total radioactivity

[0080]

[0081]

[0082] The test results are as follows: In the test sample solution, the radiochemical purity of compound 1 (%) = 100% - (1~2) (%) - 225 Actinium-DTPA (%) - No. 20 (%) - No. 28 (%) = 99.4%, Compound 1 is well separated from impurities and there is no tailing phenomenon.

[0083] Table 3 is Figure 1-5 The composition of the corresponding detection solution and the detection results

[0084]

[0085] 225 Actinium, as an alpha nuclide, emits relatively weak gamma rays. Conventional online flow detectors using sodium iodide scintillators to detect its radioactivity cannot accurately quantify these weak gamma rays. Furthermore, its complex decay chain presents significant challenges to quality control. Currently, there is no direct method for measuring its gamma rays. 225 The method of alpha decay of actinium is therefore provided by the IAEA using its daughter products. 221 Francium was measured indirectly using gamma rays.

[0086] 225Actinium, as a long-lived nuclide, has daughter nuclides that are relatively short-lived. These nuclides and daughter nuclides reach a long-term equilibrium (the concept of long-term equilibrium: the parent nucleus has a much longer half-life than the daughter nucleus, and the number of parent nuclei remains almost constant during the observation period. After reaching long-term equilibrium, the ratio of parent to daughter nuclei remains unchanged, and the radioactivity of daughter nuclei is equal to that of parent nuclei). Therefore, long-term equilibrium can be achieved through... 221 Francium signal representation 225 Actinium detection.

[0087] according to Figure 1-5 As can be seen from the results in Table 3:

[0088] For the same sample, using the IAEA-recommended trifluoroacetic acid-water and acetonitrile mobile phases and the patented method mobile phase, respectively, from... Figure 1 and Figure 3 The radiochemical purities of compound 1 were found to be 84.3% and 99.4%, respectively. This indicates that the results of the radiochemical purity test of compound 1 using trifluoroacetic acid water and acetonitrile as the mobile phase recommended by the IAEA do not match the actual sample conditions.

[0089] from Figure 1 Learned 225 Actinium-DTPA exhibits severe peak tailing under conditions of trifluoroacetic acid in water and acetonitrile, while under the conditions of the method of this invention ( Figure 4 The peaks are sharp and well separated from the main peak.

[0090] Adding approximately 5% 225 Actinium-DTPA impurities were introduced into the sample of compound 1, and their 225 The actinide-DTPA content was 6.31%. Figure 5 This indicates that the method has high accuracy and can accurately determine... 225 The true content of actinium-DTPA was determined, thus obtaining accurate radiochemical purity results for compound 1.

[0091] Example 2: Equilibrium Time Validation Test

[0092] System suitability solution: Take 95 μl of the test solution described in Example 1 and add it to approximately 5 μl of the solution described in Example 1. 225 Prepare the Actinium-DTPA impurity control solution and shake well.

[0093] The distillate of the system suitability solution was tested after being left to stand for 45 min, 3 h, 6 h, 15 h, and 24 h.

[0094] Table 4 Results of Long-Term Equilibrium Tests

[0095]

[0096] The experimental results show that the detection results of the distillate are basically consistent after equilibration for 45 min to 24 h, indicating that the detection principle of this method conforms to the long-term equilibrium principle.

[0097] Example 3 Specificity Test

[0098] Blank solution: water;

[0099] Test solution: i.e., the test solution described in Example 1;

[0100] System suitability solution: Take 95 μl of the test solution described in Example 1 and add approximately 5 μl of the solution described in Example 1. 225 Prepare the Actinium-DTPA impurity control solution and shake well.

[0101] Table 5 Specificity Test Results

[0102]

[0103] In system suitability solutions, 225 The actinium-DTPA fraction is located at position 8, and the sample peak is at position 12; in the plotted spectrum (e.g., Figure 5 ), 225 Actinium-DTPA showed good baseline separation from the test sample peak. Furthermore, neither the blank solution nor the test sample solution interfered with the detection of impurities, demonstrating the method's high specificity.

[0104] Example 4 Sensitivity Test

[0105] Background count rate: Take 10 blank liquid chromatography vials, place them in a gamma counter for detection, record their count rate (CPM, Counts Per Minute), and calculate their average value;

[0106] Limit of Quantification Solution: Take an appropriate amount of the test solution and dilute it quantitatively with water until the count rate (CPM) is more than 10 times the background count rate, and record the count rate (CPM).

[0107] Detection limit solution: Take an appropriate amount of the radionuclide solution and dilute it quantitatively with water until the count rate (CPM) is more than 3 times the background count rate, and record the count rate (CPM).

[0108] Table 6 Sensitivity Test Results

[0109]

[0110] As shown in Table 6, both the detection limit and the quantitation limit meet the IAEA sensitivity requirements (IAEA requires that the quantitation limit / detection limit should meet at least 1% of the sample concentration), thus satisfying the detection requirements.

[0111] Example 5: Linearity and Range

[0112] Take appropriate amounts of the test solution described in Example 1, and quantitatively dilute it stepwise with water to prepare linear solutions with different activities. Detect these solutions using a gamma counter and record the count rate (CPM). Plot a linear regression equation with activity (kBq) on the x-axis and count rate (CPM) on the y-axis. The results are shown in Table 7, and the obtained linear regression curves are shown in [Table 7]. Figure 7 .

[0113] Table 7. Results of Linearity and Range Tests

[0114]

[0115] Experimental results show that the method exhibits good linearity.

[0116] Example 6 Accuracy and Precision

[0117] Spiked test solution: Take 47.5 μl of the test solution described in Example 1 and add approximately 2.5 μl of the solution described in Example 1. 225 Prepare six parallel portions of the actinium-DTPA impurity control solution by shaking well.

[0118] Calculation formula:

[0119]

[0120]

[0121] The accuracy and precision results are shown in Table 8.

[0122] Table 8. Results of Accuracy and Precision Tests

[0123]

[0124] Conclusion: The experimental results show that the method has good accuracy and precision.

[0125] Example 7 Durability

[0126] Minor adjustments were made to the chromatographic condition parameters to examine the robustness of the chromatographic conditions; the specific procedures are shown in Table 9.

[0127] Table 9 Durability Test Conditions

[0128]

[0129] Take the test solution described in Example 1, perform the detection according to the above chromatographic detection conditions, record the chromatogram, and the specific experimental results are shown in Tables 10 to 12.

[0130] Table 10 Durability Test (Column Temperature) Results

[0131]

[0132] Table 11. Results of robustness tests (different batches of chromatographic columns)

[0133]

[0134] Table 12 Results of durability tests (at different flow rates)

[0135]

[0136] As can be seen from the experimental results in Tables 10 to 12, fine-tuning of the column temperature, flow rate, and chromatographic column conditions has little impact on the detection of impurities, indicating that the detection method of this invention has good robustness.

[0137] Example 8 Solution Stability

[0138] The test solution described in Example 1 was placed in a refrigerator (5 °C), and then detected at 0, 24, and 72 h. The radiochemical purity and [other components] in the test solution were determined using high-performance liquid chromatography (HPLC) at different time points. 225 The Ac-DTPA content and results are shown in Table 13.

[0139] Table 13 Stability Test Results

[0140]

[0141] The results showed that the test solution was stable at 5 °C for 72 hours.

[0142] Example 9 (Compound 1, Compound 2, 225 Actin-DOTA-TATE 225 Actinium-PSMA-617 comparison)

[0143] Preparation of PSMA-617 precursor solution: Take an appropriate amount of PSMA-617 precursor, dissolve it in water and dilute it to 1 nmol / μl;

[0144] Preparation of DOTA-TATE precursor solution: Take an appropriate amount of PSMA-617 precursor, dissolve it in water and dilute it to 1 nmol / μl;

[0145] Preparation of the precursor solution of compound 2: Take an appropriate amount of compound 2, dissolve it in water and dilute it to 1 nmol / μl;

[0146] Buffer solution preparation: Take an appropriate amount of sodium acetate, dissolve it in aqueous solution until the sodium acetate concentration is 1 mol / L, and adjust the pH to 7.0 with 1 mol / L sodium hydroxide;

[0147] Diluent stock solution: Take appropriate amounts of vitamin C and gentian acid, dissolve and dilute with water to an aqueous solution containing 281.6 mg of vitamin C and 1 mg of gentian acid per 1 ml of solution, and adjust the pH to 5.8 with 1 mol / L sodium hydroxide solution;

[0148] Diluent: Take an appropriate amount of DTPA, dissolve it by sonication with the stock solution of diluent, and dilute it to an aqueous solution containing 281.6 mg of vitamin C, 1 mg of gentian acid, and 0.2 mg of DTPA per 1 ml.

[0149] Take 30 μl of the radionuclide solution, 30 μl of the PSMA-617 precursor solution, and 360 μl of the buffer solution, react at 95 °C for 15 min, add 420 μl of diluent, and shake well to obtain the final product. 225 Actinium-PSMA-617 solution.

[0150] Take 30 μl of the radionuclide solution, 30 μl of the DOTA-TATE precursor solution, and 360 μl of the buffer solution, react at 95℃ for 15 min, add 420 μl of diluent, and shake well to obtain the final product. 225 Actinium-DOTA-TATE solution.

[0151] Take 30 μl of the radionuclide solution, 30 μl of the precursor solution of compound 2, and 360 μl of the buffer solution, react at 95℃ for 15 min, add 420 μl of diluent, and shake well to obtain the compound 2 solution.

[0152] The test results are shown in Table 14.

[0153] Table 14

[0154]

[0155] As shown in Table 14, the detection method of this application is applicable to compounds 1, 2, 3 and 4, and the detection results are highly accurate. However, using the 0.1% trifluoroacetic acid aqueous solution and acetonitrile solution recommended by IAEA as the mobile phase will result in a serious underestimation of the radiochemical purity of compounds 1 and 4, thus affecting the judgment of the labeling results.

[0156]

[0157] Compound 2

[0158]

[0159] Compound 3

[0160]

[0161] Compound 4.

Claims

1. A 225 A method for detecting radiochemical purity of an Ac conjugate, characterized by, The method employs high-performance liquid chromatography, with mobile phases including mobile phase A and mobile phase B, and gradient elution is performed. The mobile phase A mainly consists of a mixed solution of buffer salt and cation-pair reagent; The buffer salt is selected from acetates; and / or the cation-pairing reagent is selected from tetrabutylammonium salts; In the mobile phase A, the concentration of the cation-pair reagent is 2-8 mmol / L; the concentration of the buffer salt is 20-30 mmol / L. The mobile phase B comprises one or more of acetonitrile, methanol, and tetrahydrofuran; The high-performance liquid chromatography column is an octadecyl bonded silica column; With the total volume of the mobile phase being 100%, the gradient elution process includes: From 0 to 2 minutes, the volume fraction of mobile phase A is 90% to 100%, and the volume fraction of mobile phase B is 0% to 10%. Over 2-17 minutes, the volume fraction of mobile phase A decreased from 90%-100% to 30%-40%, while the volume fraction of mobile phase B increased from 0%-10% to 50%-70%. The 225 The Ac conjugate is selected from the group consisting of Compound 1, Compound 2, Compound 3, and Compound 4; The chemical formula of compound 1 is shown in Formula I: Formula I; The chemical formula of compound 2 is shown in Formula II: Formula II; The chemical formula of compound 3 is shown in Formula III: Formula III; The chemical formula of compound 4 is shown in Formula IV: Formula IV.

2. The detection method as described in claim 1, characterized in that, The buffer salt is sodium acetate; the cation-pairing reagent is tetrabutylammonium salt.

3. The detection method as described in claim 1, characterized in that, The pH of the mobile phase A is 5.5-6.

5.

4. The detection method as described in claim 1, characterized in that, The chromatographic columns used in the high-performance liquid chromatography were selected from Atlantis T3, Luna C18, Ultimate C18, and Premier Peptide CSH C18.

5. The detection method as described in claim 1, characterized in that, The flow rate of the mobile phase is 0.8-1.2 ml / min, and the column temperature of the chromatographic column is 25-35℃.

6. The detection method as described in claim 1, characterized in that, The 225 The equilibrium time for Ac is 45 min - 24 h. 221 The equilibrium time for Fr is 45 min - 24 h.

7. The detection method as described in claim 1, characterized in that, The detector in the detection method is a gamma counter.

8. The detection method as described in claim 1, characterized in that, The sampling frequency of the fraction collector in the method is 55-65 s / sample.

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