Liquid chromatographic analysis method for < 18 > F-PSMA-1007 radiochemical purity in biological sample

Abstract

The invention discloses a liquid chromatographic analysis method for < 18 > F-PSMA-1007 radiochemical purity in a biological sample. The liquid chromatographic analysis method comprises the following steps: S1, dissolving, diluting and filtering the biological sample; s2, selecting a Shim-pack GISTC18 chromatographic column, the size of the chromatographic column being 4.6 * 250 mm, and the particle size being 5 [mu] m; mobile phase selection: a mobile phase A is a mixed solution of formic acid and water, and the volume fraction of formic acid is 0.01-0.5%; a mobile phase B is acetonitrile, and gradient elution is carried out; the gradient procedure is as follows: 0-6 minutes: the mobile phase B is gradually changed from 10% to 40%; in 6-19 minutes, the mobile phase B is gradually changed from 40% to 90%; in 19-22 minutes, the proportion of the mobile phase B is maintained to be 90%; 22.1-30 minutes: maintaining the proportion of the mobile phase B to be 10%; column temperature: setting the column temperature to be 40 DEG C so as to ensure the stability of the separation effect; s3, injecting the biological sample treated in the step S1 into an HPLC (High Performance Liquid Chromatography) system to complete separation; s4, radioactivity detection: detecting radioactivity of each component of chromatographic column outflow by using a radioactivity detector, and converting collected radioactivity signals into data; and S5, data analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and specifically relates to a method for 18 Liquid chromatography analysis method for radiochemical purity of F-PSMA-1007. Background Art

[0002] In recent years, with the widespread application of prostate-specific membrane antigen (PSMA) targeted imaging in the diagnosis of prostate cancer, its sensitivity and specificity have been significantly improved, and PSMA-targeted radiopharmaceuticals have become a current research hotspot. There are many types of PSMA ligand radiolabeled nuclides commonly used in clinical practice, mainly including 68 Ga, 18F , 111 In, 11 C. 64 Cu et al., among which, 68 Ga-PSMA-11 is the most widely studied and applied PSMA tracer.

[0003] compared to 68 Ga, 18 F has more superior performance, especially in clinical applications. These advantages are mainly reflected in: 18 F has a longer physical half-life (110 minutes vs. 68 minutes), abundant production and low cost, high spatial resolution, and excellent imaging quality. 18 F-labeled PSMA ligands have become the focus of current research.

[0004] In many 18 In the F-labeled PSMA ligand, 18 F-PSMA-1007 has attracted widespread attention in the initial staging, biochemical recurrence, and subsequent follow-up studies of prostate cancer due to its unique hepatobiliary excretion properties and good tumor uptake rate.

[0005] In order to ensure the safety of medication, it is necessary to conduct relevant research in a timely and efficient manner, examine the blood concentration of drugs in humans and animals, and improve the absorption, distribution, metabolism, and excretion of drugs in the body. Accurate and rapid detection methods can further increase the accuracy of clinical trials and shorten detection time. 18 F-PSMA-1007 has innovative structure and there is no other 18 The biological sample detection and analysis method of F-PSMA-1007 therefore needs to provide an accurate, efficient, simple and rapid detection method. Summary of the invention

[0006] The purpose of the present invention is to solve the deficiencies in the prior art and provide a stable, reliable, sensitive and accurate method for detecting biological samples with a short detection cycle. 18 Liquid chromatography analysis method for radiochemical purity of F-PSMA-1007.

[0007] In order to solve the above technical problems, the technical method adopted by the present invention is as follows: the present invention discloses a method for 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 comprises the following steps:

[0008] S1. Dissolve, dilute and filter biological samples;

[0009] S2. Use Shimadzu Shim-pack GIST C18 chromatographic column with a size of 4.6×250 mm and a particle size of 5 μm;

[0010] Mobile phase selection:

[0011] Mobile phase A: a mixture of formic acid and water, with a formic acid volume fraction of 0.01% to 0.5%;

[0012] Mobile phase B: acetonitrile, gradient elution; the gradient program is as follows:

[0013] 0-6 minutes: Mobile phase B gradually changes from 10% to 40%;

[0014] 6-19 minutes: Mobile phase B gradually changes from 40% to 90%;

[0015] 19-22 minutes: Maintain the proportion of mobile phase B at 90%;

[0016] 22.1-30 minutes: maintain the proportion of mobile phase B at 10%;

[0017] Column temperature: Set the column temperature to 40°C to ensure the stability of the separation effect;

[0018] S3. Injecting the biological sample processed in step S1 into the HPLC system to complete the separation;

[0019] S4. Radioactivity detection: using a radioactivity detector to detect the radioactivity of each component outflowing from the chromatographic column, and converting the collected radioactivity signal into data;

[0020] S5. Data Analysis,By analyzing the HPLC chromatogram, the retention time of each component in the sample can be,identified, and the concentration of each component can be quantified based on,its radioactive signal intensity.

[0021] Furthermore, in step S2, the column temperature is set to 40°C.

[0022] Furthermore, in step S2, the sample loading volume is set between 20 and 100 μl.

[0023] Furthermore, in step S2, the flow rate is set to 0.8-1.2 mL / min.

[0024] Furthermore, in step S2, the injector temperature is set to 4°C.

[0025] Furthermore, the biological samples include plasma samples, urine samples and tissue samples.

[0026] Furthermore, the pretreatment steps of the plasma samples included: collecting whole blood, anticoagulating with EDTA K2, and separating plasma; taking 80 μL of plasma sample, adding 160 μL of acetonitrile to mix, centrifuging at 14000 rpm for 5 min at 4°C, taking 180 μL of supernatant, drying with N2 at 40°C, and reconstituting with 100 μL of the starting ratio mobile phase.

[0027] Furthermore, the urine sample pretreatment step includes: taking 80.0 μL of urine sample, adding 400 μL of methanol, vortexing to mix, centrifuging at 14000 g for 5 min at 2-8°C, taking 450 μL of supernatant, blowing dry with N2 at 60°C, and re-dissolving with 150 μL of the starting ratio mobile phase.

[0028] Furthermore, the tissue sample pretreatment step includes: taking the tissue sample to be tested, homogenizing it with 15 times the weight of physiological saline; taking 80.0 μl of the homogenate, adding 400 μl of methanol, vortexing and mixing, centrifuging at 14000g for 5 min at 2-8°C, taking 450 μl of the supernatant, blowing dry with N2 at 60°C, and re-dissolving with 150 μl of the starting ratio mobile phase.

[0029] Furthermore, 18 The lower limits of quantification of F-PSMA-1007 in plasma were 1.37 μCi, in urine they were 2.03 μCi, and in tissues they were 2.49 μCi.

[0030] Beneficial effects:

[0031] In the biological sample of the present invention 18 The liquid chromatography analysis method of F-PSMA-1007 radiochemical purity has significant advantages over existing designs in terms of sensitivity, detection accuracy, ease of operation, analysis speed, and specificity. Its superior performance makes it particularly suitable for 18 The application of F-PSMA-1007 in pharmacological research and clinical trials can provide reliable support for drug safety and pharmacokinetic studies.

[0032] 1. Compared with traditional HPLC-UV and LC-MS methods, this method has higher sensitivity and can detect 18 The quantitative limits of F-PSMA-1007 are as low as 1.37 μCi (plasma), 2.03 μCi (urine), and 2.49 μCi (tissue), meeting the needs of preclinical pharmacology and pharmacokinetic studies for high-sensitivity detection.

[0033] 2. This method is fast and easy to operate, using gradient elution and appropriate chromatographic conditions to ensure rapid separation and detection. The overall analysis time is short, which can significantly improve experimental efficiency and is suitable for preclinical research and clinical applications that require rapid analysis of a large number of samples.

[0034] 3. This method was verified by specificity test, and the method can clearly distinguish the target substances ( 18 F-PSMA-1007) and interferences to ensure accurate analysis. At the same time, no new radioactive peaks were detected in plasma samples placed at room temperature for 2 hours, indicating that it has good stability and is suitable for sample processing in preclinical and clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 In Example 1 of the present invention 18 The plasma Radio-HPLC spectrum of F-PSMA-1007. Among them, RT: 9.394min is 18 F-PSMA-1007.

[0036] Figure 2 In Example 2 of the present invention 18 Radio-HPLC spectrum of urine of F-PSMA-1007. RT: 9.253min is 18 F-PSMA-1007.

[0037] Figure 3 In Example 3 of the present invention 18 Radio-HPLC spectrum of F-PSMA-1007 in kidney tissue. RT: 9.339min is 18 F-PSMA-1007.

[0038] Figure 4 The Radio-HPLC spectrum of the blank solvent (starting ratio mobile phase) obtained in the specificity test of Example 4 is shown in FIG.

[0039] Figure 5 The Radio-HPLC spectrum of the free fluorine solution obtained in the specificity test of Example 4.

[0040] Figure 6The test sample obtained from the specificity test of Example 4 18 Radio-HPLC spectrum of F-PSMA-1007. RT: 9.343min is 18 F-PSMA-1007.

[0041] Figure 7 The blank plasma obtained from the specificity test of Example 4 was added 18 Radio-HPLC spectrum of F-PSMA-1007. RT: 9.347min is 18 F-PSMA-1007. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0043] The present invention proposes a method for use in plasma, urine and tissues 18 The high performance liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is stable and reliable, sensitive and accurate, with a short detection cycle and simple sample pretreatment, and is suitable for relevant experimental research on humans and animals.

[0044] The chromatographic conditions are:

[0045] Chromatographic column: Shimadzu Shim-pack GIST C18 column (4.6×250 mm, 5 μm).

[0046] Mobile Phase:

[0047] Mobile phase A: a mixed solution of formic acid and water, with a volume fraction of formic acid of 0.01-0.5%;

[0048] Mobile phase B: acetonitrile.

[0049] Gradient elution program:

[0050] 0-6 minutes: Mobile phase B gradually changes from 10% to 40%;

[0051] 6-19 minutes: Mobile phase B gradually changes from 40% to 90%;

[0052] 19-22 minutes: Maintain the proportion of mobile phase B at 90%;

[0053] 22.1-30 minutes: Maintain the proportion of mobile phase B at 10%.

[0054] Column temperature: 40℃.

[0055] Sample volume: 20-100 μL.

[0056] Flow rate: 0.8~1.2mL / min.

[0057] Injector temperature: 4°C.

[0058] Radioactivity detector parameters: energy range: 60-2000keV; smoothing time: 1 second; sensitivity: 40000cps / V; radioactivity probe: Raytest (model: Gabi).

[0059] Pretreatment of plasma samples:

[0060] 1. Collect intravenous administration 18 Whole blood of animals or humans after F-PSMA-1007 preparation, anticoagulated with EDTA K2;

[0061] 2. Centrifuge the whole blood sample at 6000 rpm for 5 minutes and collect 80 μL of plasma;

[0062] 3. Add 160 μL of acetonitrile and mix well, then centrifuge at 14,000 rpm for 5 minutes at 4°C;

[0063] 4. Take 180 μL of supernatant and blow dry with nitrogen (N2) at 40°C;

[0064] 5. Reconstitute with 100 μL of starting ratio mobile phase.

[0065] Pretreatment of urine samples:

[0066] 1. Take the medicine through the vein 18 80 μL of animal urine after F-PSMA-1007 formulation;

[0067] 2. Add 400 μL methanol and vortex to mix;

[0068] 3. Centrifuge at 14000g for 5 minutes at 2-8°C;

[0069] 4. Take 450 μL of supernatant and blow dry with nitrogen at 60°C;

[0070] 5. Reconstitute with 150 μL of starting ratio mobile phase.

[0071] Pre-treatment of tissue samples:

[0072] 1. Take tumor, liver, kidney, heart, muscle and other tissues and homogenize them with 15 times the weight of normal saline;

[0073] 2. Take 80 μL of homogenate, add 400 μL of methanol, and vortex to mix;

[0074] 3. Centrifuge at 14000g for 5 minutes at 2-8°C;

[0075] 4. Take 450 μL of supernatant and blow dry with nitrogen at 60°C;

[0076] 5. Reconstitute with 150 μL of starting ratio mobile phase.

[0077] Detection limit:

[0078] In plasma 18 The lower limit of quantification of F-PSMA-1007 was 1.37 μCi;

[0079] In urine 18 The lower limit of quantification of F-PSMA-1007 was 2.03 μCi;

[0080] In the organization 18 The lower limit of quantification of F-PSMA-1007 was 2.49 μCi.

[0081] Scope of application:

[0082] The method of the present invention is applicable to the blood, urine and tissues of humans and animals (such as rats, mice, beagles, Bama miniature pigs, etc.) 18 Radiochemical purity analysis of F-PSMA-1007.

[0083] Example:

[0084] Example 1: Plasma 18 Analytical Method for Radiochemical Purity of F-PSMA-1007

[0085] Sample: Collected intravenously 18 F-Plasma of SD rats after PSMA-1007 formulation;

[0086] Chromatographic conditions: The chromatographic column was Shimadzu Shim-pack GIST C18 column (4.6×250 mm, 5 μm);

[0087] Mobile phase A is a mixed solution of formic acid and water, with a volume fraction of formic acid of 0.1%;

[0088] The mobile phase B is acetonitrile, and gradient elution is performed, wherein the gradient elution program is as follows: within 0 to 6 minutes, 10% of the mobile phase B gradually changes to 40% of the mobile phase B;

[0089] Within 6 to 19 minutes, 40% mobile phase B gradually changed to 90% mobile phase B;

[0090] From 19 to 22 minutes, the proportion of mobile phase B was maintained at 90%;

[0091] 22. From 1min to 30min, the proportion of mobile phase B is maintained at 10%;

[0092] Column temperature: 40°C; sample volume: 100 μl; flow rate: 1.0 ml / min; injector temperature: 4°C.

[0093] Radioactive detector parameters: Energy: 60-2000keV; Smooth: 1s; Range: 40000cps / V. Radioactive probe: Raytest (model: Gabi);

[0094] Samples to be tested: Plasma to be tested is intravenously administered 18F -PSMA-1007-treated SD rats' whole blood was anticoagulated with EDTA-K2 and the separated plasma was prepared for analysis immediately after collection.

[0095] Preparation steps of the test sample: 80 μl of plasma was added with 160 μl of acetonitrile and mixed, centrifuged at 14000 rpm for 5 min at 4°C, and 180 μl of the supernatant was heated at 40°C for 1 h. 2 Blow dry, reconstitute with 100 μl of the starting ratio mobile phase, and take the supernatant.

[0096] Specific steps of sampling: Take blank solvent (starting proportion mobile phase) and test sample for sampling respectively, confirm that the method is specific, take the treated sample for sampling, and use the normalization method to calculate the plasma 18F - Radiochemical purity of PSMA-1007, investigation 18F -Metabolism of PSMA-1007 in SD rats.

[0097] Typical spectra Figure 1 . Figure 1 for 18F -PSMA-1007 plasma Radio-HPLC spectrum. RT: 9.394min is 18F -PSMA-1007. Indicates that 18 F-PSMA-1007 is of high purity and clearly separated.

[0098] Example 2: Urine 18 Analytical Method for Radiochemical Purity of F-PSMA-1007

[0099] The sample to be tested is a urine sample to be tested: intravenous administration 18F - Urine from SD rats after PSMA-1007 administration was prepared and analyzed immediately after collection.

[0100] The chromatographic conditions were the same as in Example 1.

[0101] Typical spectra Figure 2 . Figure 2 for 18F-PSMA-1007 urine Radio-HPLC spectrum. RT: 9.253min is 18F -PSMA-1007. Indicated in urine 18 F-PSMA-1007 also has good separation effect and its peak is stable.

[0102] Example 3: Kidney tissue 18 Analytical Method for Radiochemical Purity of F-PSMA-1007

[0103] The sample to be tested is kidney tissue: intravenous administration 18F - Kidneys of SD rats after PSMA-1007 administration were collected and prepared for analysis immediately after collection.

[0104] Preparation steps of the test sample: take the kidney to be tested, homogenize it with 15 times the weight of normal saline, take 80.0 μl of the homogenate, add 400 μl of methanol, vortex mix, centrifuge at about 14000g for 5 min at 2-8℃, take 450 μl of the supernatant and heat it at 60℃ for 10 min. 2 After drying, reconstitute with 150 μl of the starting ratio mobile phase, and take 100 μl for analysis by Radio-HPLC.

[0105] The chromatographic conditions were the same as in Example 1.

[0106] Typical spectra Figure 3 . Figure 3 for 18F -PSMA-1007 kidney tissue Radio-HPLC spectrum. RT: 9.339min is 18F -PSMA-1007, the peak position is still relatively stable, proving that this method is applicable to various tissue samples.

[0107] Example 4: Specificity test

[0108] Take blank solvent, free fluorine solution, 18F -PSMA-1007 formulation solution and blank plasma 18F -PSMA-1007 solution, each in appropriate amount, and the feces are injected into the liquid chromatograph, and the chromatogram is recorded. The results are shown in Figures 4 to 7 The results showed that the matrix in blank solvent, free fluoride solution and blank plasma did not interfere with 18F The determination of PSMA-1007 showed that the method had good specificity.

[0109] Example 5: Repeatability test

[0110] The same operator repeatedly prepared 6 portions of spiked test solution, injected the samples respectively under the chromatographic conditions of Example 1, and recorded the chromatograms. The results are shown in Table 1.

[0111]

[0112] The average value of 271684 represents the number of replicates. 18 The central trend of the corrected peak area for F-PSMA-1007 indicated that the measured values ​​for most samples were relatively close to this value.

[0113] The RSD in the table is 6.35%, which means that the variation of all 6 experimental measurement results is relatively small. Generally, an RSD value of less than 10% is considered to be a sign that the experimental results have good repeatability and stability. Therefore, an RSD of 6.35% indicates that this method has high stability and reliability in repeatability testing.

[0114] The data in the table show that this method can stably and reliably detect 18 F-PSMA-1007, and the measurement error is small, suitable for high-precision analysis tasks. The RSD value is 6.35%, which further verifies the high repeatability of the experiment and the reliability of the method.

[0115] Example 6: Plasma stability test

[0116] After adding the control substance to the plasma sample, it was placed at room temperature for 2 hours. Compared with 0 hours, no additional radioactive peak was found, indicating that 18 F-PSMA-1007 has good stability in plasma at room temperature.

[0117] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. In a biological sample 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: The steps include: S1. Dissolve, dilute and filter biological samples; S2. Use Shimadzu Shim-pack GIST C18 chromatographic column with a size of 4.6×250 mm and a particle size of 5 μm; Mobile phase selection: Mobile phase A: a mixture of formic acid and water, with a formic acid volume fraction of 0.01% to 0.5%; Mobile phase B: acetonitrile, gradient elution; the gradient program is as follows: 0-6 minutes: Mobile phase B gradually changes from 10% to 40%; 6-19 minutes: Mobile phase B gradually changes from 40% to 90%; 19-22 minutes: Maintain the proportion of mobile phase B at 90%; 22.1-30 minutes: maintain the proportion of mobile phase B at 10%; Column temperature: Set the column temperature to 40°C to ensure the stability of the separation effect; S3. Injecting the biological sample processed in step S1 into the HPLC system to complete the separation; S4. Radioactivity detection: using a radioactivity detector to detect the radioactivity of each component outflowing from the chromatographic column, and converting the collected radioactivity signal into data; S5. Data Analysis,By analyzing the HPLC chromatogram, the retention time of each component in the sample can be,identified, and the concentration of each component can be quantified based on,its radioactive signal intensity.

2. The biological sample according to claim 1 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: In step S2, the column temperature is set to 40°C.

3. The biological sample according to claim 2 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: In step S2, the sample loading volume is set between 20 and 100 μl.

4. The biological sample according to claim 2 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: In step S2, the flow rate is set to 0.8-1.2 mL / min.

5. The biological sample according to claim 2 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: In step S2, the injector temperature is set to 4°C.

6. The biological sample according to claim 1 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: The biological samples include plasma samples, urine samples and tissue samples.

7. The biological sample according to claim 6 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: The pretreatment steps of plasma samples included: collecting whole blood, anticoagulating with EDTA K2, and separating plasma; taking 80 μL of plasma sample, adding 160 μL of acetonitrile to mix, centrifuging at 14000 rpm for 5 min at 4°C, taking 180 μL of supernatant, blowing dry with N2 at 40°C, and reconstituted with 100 μL of the starting ratio mobile phase.

8. The biological sample according to claim 6 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: The urine sample pretreatment steps include: taking 80.0 μl of urine sample, adding 400 μl of methanol, vortex mixing, centrifuging at 14000 g for 5 min at 2-8°C, taking 450 μl of supernatant, blowing dry with N2 at 60°C, and re-dissolving with 150 μl of starting ratio mobile phase.

9. The biological sample according to claim 6 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: The tissue sample pretreatment step includes: taking the tissue sample to be tested, homogenizing it with 15 times the weight of physiological saline; taking 80.0 μl of the homogenate, adding 400 μl of methanol, vortex mixing, centrifuging at 14000g for 5 minutes at 2-8°C, taking 450 μl of the supernatant, blowing dry with N2 at 60°C, and re-dissolving with 150 μl of the starting ratio mobile phase.

10. The biological sample according to claim 6 18 The liquid chromatography analysis method for radiochemically pure F-PSMA-1007 is characterized by: 18 The lower limits of quantification of F-PSMA-1007 in plasma were 1.37 μCi, in urine they were 2.03 μCi, and in tissues they were 2.49 μCi.

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