A fluorine 18 Quality Control Method of Pyramide Injection

Abstract

The present invention provides a fluorine [ 18 The quality control method of pyramide injection adopts high performance liquid chromatography, adopts C18 chromatographic column, 10mM phosphate buffer with pH 6-8 as mobile phase A, acetonitrile as mobile phase B, and performs gradient elution. The quality control method provided by the present invention can effectively separate fluorine [ 19 F] pyramide and other UV impurities, effectively separating fluorine [ 18 F] pyramide and other radiochemical impurities provide the basis for accurate detection results; the use of ultraviolet detectors combined with radioactive flow detectors can simultaneously achieve fluorine [ 18 F]pyramide radiochemical purity and fluorine [ 19 F] determination of pyramide content and impurities; the test is completed in 20 minutes, achieving fluorine [ 18 F] Rapid test and release of pyramide injection; using labeled synthetic mother solution as impurity test sample and / or radiochemical purity spike solution to ensure that all impurities in the injection can be detected and the detection accuracy is guaranteed.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, in particular to a fluoro[ 18 F] Quality control method for pyramide injection. Background Art

[0002] fluorine[ 18 Fluoro[F]pyramide injection is a positron emission tomography (PET) radiopharmaceutical that is clinically used for the diagnosis of melanoma patients or suspected patients who express melanin. 18 F]Pyramide is the active ingredient in injection products. Its proportion of radioactive substances in the injection, i.e., its radiochemical purity, is a key quality indicator of injection products. At present, the detection method of radiochemical purity of radioactive drugs is still mainly radioactive thin layer chromatography, which is usually unable to separate and quantitatively determine impurities with similar structures (including radiochemical impurities and organic impurities).

[0003] fluorine[ 18 F]pyramide injection is a precursor compound that reacts with radioactive fluorine in a synthesizer. 18 F] ions are prepared by nucleophilic substitution to obtain fluorine [ 18 F] Pyramide, the synthetic route is shown below.

[0004]

[0005] fluorine[ 18 F]pyramide Synthesis Route

[0006] During the preparation of injection, a variety of radioactive impurities and organic impurities will be generated. Most of the impurities can be removed by separation and purification. 19 F] pyramide is fluoro[ 18 The content of stable isotope compounds of F]pyramide is also one of the factors affecting the quality of drugs, and accurate determination of its content is also a necessary quality control item. Summary of the invention

[0007] In order to accurately measure the fluorine 19 F] pyramide content and fluorine [ 18 F] pyramide radiochemical purity, the first object of the present invention is to provide a fluoro[ 18 F]pyramide injection quality control method, to achieve the fluorine [ 19 F] pyramide and other UV impurities, fluoro[ 18 F] pyramide and other radiochemical impurities, for the efficient separation of fluoro[ 19 F] pyramide content and fluorine [ 18 The present invention provides a basis for the accurate and simultaneous determination of the radiochemical purity of [F]pyramide.

[0008] In some embodiments of the present invention, the quality control method comprises using high performance liquid chromatography, wherein the high performance liquid chromatography uses a chromatographic column with C18 (octadecylsilane bonded silica gel) as a filler, uses a 10 mM phosphate buffer with a pH of 6-8 as a mobile phase A, and uses acetonitrile as a mobile phase B, and elutes according to the following gradient (wherein the percentages of mobile phase A and mobile phase B are all volume percentages):

[0009] 0-7.5 min, 70-90% mobile phase A and 10-30% mobile phase B;

[0010] 7.5-10 min, 60-80% mobile phase A and 20-40% mobile phase B;

[0011] 10-14.9 min, 20-30% mobile phase A and 70-80% mobile phase B;

[0012] 14.9-15min, mobile phase A increased from 20-30% to 70-90% and mobile phase B decreased from 70-80% to 10-30%;

[0013] 15-20 min, 70-90% mobile phase A and 10-30% mobile phase B.

[0014] The quality control method provided by the present invention adopts the mobile phase A and the mobile phase B, and performs elution according to the gradient, so as to achieve the separation of ultraviolet impurities and fluorine in the injection solution. 19 F] pyramide can be effectively separated from radiochemical impurities and fluorine [ 18 The effective separation of F]pyramide provides a basis for further testing and, based on the relevant guidelines of ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use), determines whether further control or reporting of impurities in drugs is required to ensure the safety and effectiveness of drugs.

[0015] In some embodiments of the present invention, the mobile phase A and the mobile phase B are eluted according to the following gradient (wherein the usage percentages of the mobile phase A and the mobile phase B are all volume percentages):

[0016] 0-7.5 min, 75-85% mobile phase A and 15-25% mobile phase B;

[0017] 7.5-10 min, 70-80% mobile phase A and 20-30% mobile phase B;

[0018] 10-14.9 min, 15-25% mobile phase A and 75-85% mobile phase B;

[0019] 14.9-15min, mobile phase A increased from 15-25% to 75-85% and mobile phase B decreased from 75-85% to 15-25%;

[0020] 15-20 min, 75-85% mobile phase A and 15-25% mobile phase B.

[0021] The above-mentioned specific mobile phase elution gradient can achieve the separation of ultraviolet impurities and fluorine [ 19 F] pyramide can be effectively separated from radioactive impurities and fluorine in injection solution. 18 The effective separation of F]pyramide can also achieve more efficient detection, and the fluoro[ 18 F] Testing of pyramide injection.

[0022] In some embodiments of the present invention, the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

[0023] In some embodiments of the present invention, the column temperature of the chromatographic column is 25-35°C.

[0024] In some embodiments of the present invention, the mobile phase A and the mobile phase B are eluted at a flow rate of 0.8-1.2 mL / min, preferably at a flow rate of 1.0 mL / min.

[0025] In some embodiments of the present invention, the liquid chromatography method uses an ultraviolet detector to detect fluorine [ 19 F] pyramide and / or its content, preferably detected at a wavelength of 254 nm.

[0026] The second object of the present invention is to provide a quality control method that can simultaneously achieve fluorine [ 18 Fluoride in pyramide injection 19 F] pyramide and / or its content and fluorine [ 18 The radiochemical purity of F]pyramide was further determined by the aforementioned quality control method combined with a radioactive flux detector to detect fluorine [ 18 The radiochemical purity of F]pyramide is achieved by combining the high performance liquid chromatography (ultraviolet detector) and the radioactive flow detector to simultaneously determine the fluorine [ 18 The radiochemical purity of F]pyramide and the fluorine content of injection 19 F] Pyramide content.

[0027] The third object of the present invention is to ensure that fluorine [ 18 F] All impurities in pyramide injection can be detected. On the basis of the above scheme, the labeled synthetic mother solution is used as the impurity test sample and / or the radiochemical purity spike solution to ensure the specificity and accuracy of the method.

[0028] The present invention uses high performance liquid chromatography (ultraviolet detector) combined with radioactive flow detector (Radio-HPLC) technology to simultaneously achieve radiochemical purity and fluorine content of injection products. 19 F] pyramide content and all impurities are determined simultaneously; the test can be completed in 20 minutes, achieving fluorine [ 18 F] Rapid inspection and release of pyramide injection to meet the requirements for release inspection of radioactive drugs before clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the 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 creative work.

[0030] Figure 1 The fluorine provided by the present invention 18 F] One of the UV chromatograms of the blank solution involved in the quality control method of pyramide injection.

[0031] Figure 2 The fluorine provided by the present invention 18 F] One of the UV spectra of the reference solution involved in the quality control method of pyramide injection.

[0032] Figure 3 The fluorine provided by the present invention 18 F] The quality control method of pyramide injection involves checking the UV spectrum of the reference solution.

[0033] Figure 4 The fluorine provided by the present invention 18 Fluorine [F]pyramide injection quality control method involved 18 F] One of the UV spectra of the pyramide test sample.

[0034] Figure 5 The fluorine provided by the present invention 18 Fluorine [F]pyramide injection quality control method involved 18 One of the radioactive spectra of the [F]pyramide test sample.

[0035] Figure 6 The fluorine provided by the present invention 18 F] One of the UV spectra of the impurity sample involved in the quality control method of pyramide injection.

[0036] Figure 7 The fluorine provided by the present invention18 One of the radioactive spectra of the impurity sample involved in the quality control method of F]pyramide injection.

[0037] Figure 8 The fluorine provided by the present invention 18 F] One of the UV spectra of the sample subjected to 4-hour high temperature degradation involved in the quality control method of pyramide injection.

[0038] Fig. 9 The fluorine provided by the present invention 18 One of the radioactive spectra of the test sample after 4 hours of high temperature degradation involved in the quality control method of F]pyramide injection.

[0039] Fig.10 The fluorine provided by the present invention 18 Fluorine [F]pyramide injection quality control method involved 19 F] One of the UV spectra of the quantification limit solution of pyramide.

[0040] Fig.11 The fluorine provided by the present invention 18 Fluorine [F]pyramide injection quality control method involved 18 One of the linear plots of pyramide activity.

[0041] Fig.12 The fluorine provided by the present invention 18 Fluorine [F]pyramide injection quality control method involved 19 F] One of the linear plots of pyramide content.

[0042] Fig.13 The fluorine provided by the present invention 18 F] One of the ultraviolet spectra involved in comparative example 1 of the quality control method for pyramide injection.

[0043] Fig.14 The fluorine provided by the present invention 18 F] One of the radioactive chromatograms involved in Comparative Example 2 of the quality control method for pyramide injection.

[0044] Fig.15 The fluorine provided by the present invention 18 F] One of the ultraviolet spectra involved in comparative example 2 of the quality control method for pyramide injection.

[0045] Fig.16 The fluorine provided by the present invention 18 F] One of the radioactive chromatograms involved in Comparative Example 3 of the quality control method for pyramide injection.

[0046] Fig.17 The fluorine provided by the present invention 18F] One of the ultraviolet spectra involved in Example 3 of the quality control method for pyramide injection. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Chemical drug preparations are usually composed of active ingredients and excipients. The detection of the content or purity of active ingredients in preparations usually gives priority to the analytical method of active ingredients. 18 F] Labeled drugs are usually obtained from precursor compounds through chemical reactions, separation and purification, and mixing with excipients to obtain the final injection product.

[0049] The active ingredient fluorine in the injection solution of the present invention is partly involved in the embodiments 18 F] Pyramide is obtained by labeling with a precursor compound having the structural formula shown below.

[0050]

[0051] In the labeling reaction, the p-toluenesulfonic acid group TsO- of the precursor compound leaves and is replaced by fluorine [ 18 F] ion substitution to generate the radioactive active ingredient of the preparation [ 18 F]pyramide, at the same time, a small amount of stable fluorine introduced by reaction reagents, equipment pipelines, etc. 19 F] ions react with some precursor compounds to form stable fluorine [ 19 F] pyramide. The precursor compound is labeled to generate fluoro[ 18 The solution after the labeling reaction of pyramide injection is called the labeling synthesis mother solution. The labeling synthesis mother solution contains fluorine [ 18 F]pyramide, also rich in precursor compounds, fluoro[ 19 F]pyramide and other impurities, such as compounds containing fluorine [ 18 F] radiochemical impurities, organic process impurities and degradation impurities. Among them, fluorine [ 19 The content of F]pyramide needs to be controlled to avoid its interaction with radioactive fluorine [ 18 F] Competitive effects of pyramide in targeting lesions.

[0052] In the prior art, there is a method for separating fluorine by using trifluoroacetic acid aqueous solution and trifluoroacetic acid acetonitrile solution. 19 F]pyramide or fluoro[ 18F] pyramide, but the inventors found that fluoro[ 18 F]pyramide and fluoro[ 19 F]pyramide is unstable in an acidic environment. Therefore, using an aqueous solution of trifluoroacetic acid or an acetonitrile solution as the mobile phase for elution will result in the formation of fluorine [ 18 F] pyramide decomposes, affecting the accuracy of the test data. The main component to be tested is weakly retained on the C18 column. If the above mobile phase is used, the analysis time must be extended, which is not conducive to the inspection of radioactive drugs. In addition, the method does not conduct method applicability and specificity investigation on the detection of potential impurities, and cannot guarantee the effective identification and separation of impurities with similar structures. For example, the RRT 0.92 impurity in this method has the following structure (the F element is 18 F or 19 F corresponds to the radiochemical impurities or ultraviolet impurities with this chemical structure in the preparation).

[0053]

[0054] From the analysis of chemical structure, the precursor compound and fluorine [ 19 F]pyramide or fluoro[ 18 F]pyramide has little difference, so the preparation analysis method can be developed based on the precursor analysis method, and the residue of precursor compound in the preparation can be detected at the same time.

[0055] Therefore, the fluorine provided by the present invention [ 18 The development of the quality control method for [F]pyramide injection was based on the detection method of precursor compounds, and the initial mobile phase ratio, elution gradient, running time and other conditions were further optimized. The impurity-rich reaction mother liquor (labeled synthesis mother liquor) was also used for fluorine [ 19 F]pyramide and fluoro[ 18 F]pyramide test conditions were screened, representative optimized conditions were selected, and the quality control method provided by the present invention was obtained to achieve the quality control of fluoro[ 18 F]pyramide injection radiochemical purity and fluorine [ 19 F] The pyramide content and the detection of various actual impurities in the labeled synthesis mother liquor ensure that all impurities can be detected and the analysis time is shortened as much as possible, and the detection can be completed in just 20 minutes.

[0056] The quality control method provided by the present invention is that the ultraviolet detector is connected in series with the radiation flow detector. When performing the detection, the sample flows through the chromatographic column and is eluted by the mobile phase. The eluent first flows through the ultraviolet detector to perform fluorine [ 19 F]pyramide and UV impurities, and then the eluent flows through a radiometric flow detector for fluorine [ 18 F] Detection of radiochemical purity of pyramide and radiochemical impurities.

[0057] The information of reagents, instruments, etc. used in the examples of the present invention are shown in the following table.

[0058]

[0059] In the embodiments of the present invention, unless otherwise specified, the solutions involved are prepared as follows:

[0060] Blank solution: 0.9% sodium chloride solution;

[0061] Test solution: Fluorine [ 18 F] Pyramide injection;

[0062] Reference solution (100% level): Accurately weigh about 20 mg of fluorine 19 F] pyramide reference substance was placed in a 100 mL volumetric flask and dissolved in acetonitrile to prepare fluoro[ 19 F] pyramide reference stock solution (stored at -20°C); take 2.0 mL of the reference stock solution and place it in a 20 mL volumetric flask, add 0.9% sodium chloride solution to dilute to volume, and obtain fluoro[ 19 F] Pyramide reference solution, content is about 20μg / mL.

[0063] Sensitivity solution: Accurately measure 1 mL of the reference solution and place it in a 100 mL volumetric flask. Dilute to volume with the blank solution and shake well to obtain a concentration of about 0.2 μg / mL (1% level).

[0064] Mobile phase A: Accurately weigh 0.540 g of sodium dihydrogen phosphate (anhydrous) and 0.979 g of disodium hydrogen phosphate (dihydrate), dissolve in water and make up to 1000 mL, and degas by ultrasonication for 15 minutes.

[0065] Mobile phase B: Take an appropriate amount of acetonitrile and degas by ultrasonication for 15 minutes.

[0066] The above solutions can be prepared in volumes that are adjusted according to dosage while maintaining the target concentration.

[0067] Example 1

[0068] This embodiment provides a fluorine [ 18 F]pyramide injection quality control method, including simultaneous detection of fluorine [ 18 F]pyramide radiochemical purity and its fluorine [ 19 F] The content of pyramide was determined by high performance liquid chromatography-radioactive flow detector combined method.

[0069] The conditions of high performance liquid chromatography are:

[0070] Chromatographic column: C18 column (4.6 mm × 250 mm, 5 μm);

[0071] Mobile phase A: 10 mM phosphate buffer, pH 7.0;

[0072] Mobile phase B: acetonitrile;

[0073] Gradient elution program:

[0074]

[0075] Flow rate: 1.0 mL / min;

[0076] Column temperature: 30°C;

[0077] UV detector wavelength: 254nm;

[0078] Injection volume: 10 μL;

[0079] Running time: 20 minutes.

[0080] First, the reliability of the quality control method provided in this embodiment is verified.

[0081] 1. System applicability

[0082] Blank solution: 0.9% sodium chloride solution;

[0083] Reference solution: 22.20 μg / mL fluoride 19 F] Pyramide reference solution;

[0084] Verification reference solution: 22.48 μg / mL fluoride 19 F] Pyramide reference solution;

[0085] Test solution: 20240704 batch of fluorine [ 18 F] Pyramide injection.

[0086] After the HPLC baseline is stable, inject samples in the following order: inject at least 1 injection of blank solution (i.e. 10 μL), inject 6 injections of reference solution in succession, inject 1 injection of verification reference solution, and inject 1 injection of fluorine [ 18 F] The pyramide test solution was injected 6 times continuously, and the UV signal and radioactive signal were recorded respectively, and the chromatogram was saved, such as Figure 1-5 shown.

[0087] from Figure 1-Figure 5It can be concluded that the blank solution has no interference at the main ultraviolet peak of the reference solution; the relative standard deviation of the peak area of ​​the main peak of the reference solution for 6 consecutive injections is 0.04% (satisfying ≤2%), and the relative standard deviation of the retention time is 0.11% (satisfying ≤1%); the recovery rate of the reference solution was verified to be 100.5% (satisfying 95~105%); in the radioactivity spectrum, the relative standard deviation of the retention time of the main radioactive peak of the 6 injections of the test solution was 0.08% (satisfying ≤1.0%), and the relative standard deviation of the radiochemical purity was 0.05% (satisfying ≤1.0%).

[0088] The above results indicate that the quality control method provided in Example 1 has passed the system applicability and has good injection precision.

[0089] 2. Exclusive inspection

[0090] (1) Solution preparation

[0091] Blank solution: 0.9% sodium chloride solution;

[0092] Test solution: 20240704 batch of fluorine [ 18 F] Pyramide injection;

[0093] Reference solution: 22.20 μg / mL fluoride 19 F] Pyramide reference solution;

[0094] Specific spiked sample: Mix the test solution and the reference solution in equal volume proportions to obtain;

[0095] High temperature degradation sample: Take the specific spiked sample and place it in a 60℃ stabilization box for 4 hours;

[0096] Impurity test sample: 20240704 batch of fluorine [ 18 F] The mother liquor rich in impurities after the labeling reaction of pyramide injection (i.e., the labeled synthesis mother liquor). Using the labeled synthesis mother liquor as the impurity test sample can ensure that all potential impurities can be detected.

[0097] (2) Operation

[0098] After the HPLC baseline is stable, inject at least 1 injection of blank solution, 6 injections of reference solution, 1 injection of specific spiked sample, 1 injection of impurity sample, and 1 injection of high temperature degradation sample. Record the UV signal and radioactive signal respectively and save the chromatogram. The UV spectrum and radioactive spectrum of the impurity sample are as follows: Figure 6 and Figure 7 As shown, Figure 7 The 6.835min is fluorine [ 18 The peak of pyramide is at 2.425min, which is free fluorine [ 18F] ion peak, and other peaks are other radiochemical impurity peaks; Figure 6 The 6.843min is fluorine [ 19 The peak of F]pyramide is at 13min, the peak is the precursor compound, and the others are UV chemical impurity peaks; the UV spectrum and radioactive spectrum of the test sample after high temperature degradation for 4 hours are as follows Figure 8 and Fig. 9 As shown, fluorine [ 18 The peak of [F]pyramide is at 6.831min ( Figure 8 The other peaks are radiochemical impurity peaks), fluorine [ 19 The peak of [F]pyramide is at 6.839 min.

[0099] (3) Acceptable standards

[0100] In the UV spectrum of the specific spiked sample, fluorine [ 19 The ratio of the retention time of pyramide to the average retention time of the main peak of the reference solution should be between 0.98 and 1.02; the impurity test sample and the fluorine in the high temperature degradation sample should be between 0.98 and 1.02. 18 The resolution between the pyramide peak and the adjacent impurity peak should be ≥1.5, and the fluorine [ 19 The resolution between the pyramide peak and the adjacent impurity peak should be ≥1.5.

[0101] (4) Results: As shown in Table 1.

[0102] Table 1 Specificity verification results

[0103]

[0104] It can be seen from Table 1 that fluorine [ 19 The ratio of the retention time of pyramide to the average retention time of the main peak of the reference solution was 1.00 (satisfying the requirement of 0.98-1.02); the impurity test sample and the fluorine in the high temperature degradation sample 18 The resolution between the pyramide peak and the adjacent impurity peaks was greater than 1.5, and the fluorine [ 19 F] The separation degree between the pyramide peak and the adjacent impurity peak is greater than 1.5, indicating that the specificity of the quality control method provided in this example has been verified, the method has good specificity, and the component to be measured is not interfered by the matrix component.

[0105] 3. Limit of Quantitation

[0106] (1) Solution preparation

[0107] Blank solution: 0.9% sodium chloride solution;

[0108] Test solution: Fluorine [ 18 F] Pyramide injection;

[0109] fluorine[19 F] Pyramide quantification limit solution: sensitivity solution, prepared in parallel by diluting the reference solution in 6 portions;

[0110] Activity limit of quantitation solution: The lowest concentration specification of injection solution (10mCi / mL) has a radioactive concentration of 0.23mCi / mL in 10 hours. The injection volume is 10μl, and the lowest activity of the injection is 2.3μCi. The activity limit of quantitation should be lower than 2.3μCi. 18 F] Pyramide injection (test solution), calculate the radioactivity concentration at the time of injection according to the half-life and injection order, dilute with blank solution to the expected radioactivity concentration, and prepare 6 activity quantitation limit solutions.

[0111] (2) Operation

[0112] After the HPLC baseline is stable, take 6 portions of the activity limit solution and inject one injection in sequence. Save the radioactive chromatogram (omitted) for evaluating the activity limit of quantification. Inject 6 injections of the sensitivity solution (i.e., fluorine [ 19 F] pyramide quantitative limit solution), save the UV spectrum, such as Fig.10 As shown, it is used to evaluate the fluorine [ 19 F] Pyramide limit of quantification, Fig.10 In the 6.839min, the fluorine 19 F] Pyramide peak appears, and the solvent peak is around 18min.

[0113] (3) Acceptable standards

[0114] In the radioactive chromatogram, the activity quantification limit solution contains fluorine [ 18 The signal-to-noise ratio (S / N) of the pyramide peak should be ≥10. The injection activity is calculated based on the decay. The relative standard deviation of the activity values ​​of the six injections should be ≤20%. The quantitative limit activity level should be reported;

[0115] In the UV spectrum of sensitive solution, fluorine [ 19 The signal-to-noise ratio (S / N) of the pyramide peak should be ≥10; the relative standard deviation of the 6-needle peak area should be ≤20.0%, and the quantitative limit concentration level should be reported.

[0116] (4) Results: As shown in Table 2.

[0117] Table 2 Fluorine 18 F]pyramide activity and fluorine [ 19 F] Pyramide content quantitative limit verification results

[0118]

[0119] It can be seen from Table 2 that the average activity measured by the activity quantification limit solution is 2.7μCi, and the average signal-to-noise ratio is 246; 19The quantification limit of the [F]pyramide content was 0.22 μg / mL, and the average signal-to-noise ratio was 83.

[0120] 4. Detection limit

[0121] (1) Solution preparation

[0122] Blank solution: 0.9% sodium chloride solution;

[0123] fluorine[ 19 F] Pyramide detection limit solution: Accurately measure 5 mL of the sensitivity solution and dilute it to 10 mL with the blank solution to obtain the detection limit solution;

[0124] (2) Operation

[0125] After the HPLC baseline was stable, three injections of the detection limit solution were injected continuously and the UV spectrum (omitted) was saved for the evaluation of fluorine [ 19 F] Pyramide detection limit.

[0126] (3) Acceptable standards

[0127] In the UV spectrum of the detection limit solution, fluorine [ 19 The signal-to-noise ratio (S / N) of the [F]pyramide peak was ≥3, and the detection limit concentration level was reported.

[0128] (4) Results: As shown in Table 3.

[0129] Table 3 Fluorine 19 F]Pyramide detection limit test results

[0130]

[0131] As can be seen from Table 3, the quality control method provided in this embodiment, fluorine [ 19 The detection limit of F]pyramide content was 0.11 μg / mL.

[0132] 5. Linear range

[0133] (1) Solution preparation

[0134] Blank solvent: 0.9% sodium chloride solution;

[0135] Activity linearity test solution: Take fluorine [ 18 F] Pyramide injection was diluted with blank solution. The sample information is shown in Table 4.

[0136] Table 4 Activity linearity test product information (July 5, 2024)

[0137]

[0138] fluorine[ 19F] Pyramide linear test solution: accurately weigh 22.48 mg of fluoro[ 19 F] Pyramide reference substance is placed in a 100 mL volumetric flask, dissolved in acetonitrile and fixed to volume to prepare the reference substance stock solution. Accurately measure the reference substance stock solution and dilute it with the blank solution to prepare the linear test solution, as shown in Table 5.

[0139] Table 5 Fluorine 19 F] Pyramide Linear Test Product Information

[0140]

[0141] (2) Operation

[0142] After the baseline of the HPLC is stable, according to the specified chromatographic conditions, take at least 1 injection of blank solution and 1 injection of each radiochemical purity linear test solution, save the chromatogram (omitted), use radioactivity as the horizontal axis and peak area as the vertical axis, draw the standard curve, calculate the regression equation, correlation coefficient, and list the residual sum of squares. The theoretical value of radioactivity is calculated after the calibration activity is decay-corrected.

[0143] Take blank solution and inject at least 1 injection. 19 [F] One injection of each pyramide linear test solution was performed, and the peak area was used as the horizontal axis and the injection concentration as the vertical axis to draw the standard curve, calculate the regression equation, correlation coefficient, and list the residual sum of squares.

[0144] (3) Acceptable standards

[0145] List the linear equation; linear correlation coefficient R≥0.99; report the residual sum of squares;

[0146] Activity linear range: Report the activity from the limit of quantitation to the maximum value of the linear activity;

[0147] fluorine[ 19 F] Linear range of pyramide content: Report the concentration from the limit of quantitation to the highest linear concentration and concentration level.

[0148] (4) Results

[0149] fluorine[ 18 The linear equation and linear graph of the activity of F]pyramide are shown in Table 6 and Fig.11 As shown; Fluorine [ 19 F] The linear equation and linear graph of pyramide content are shown in Table 7 and Fig.12 shown.

[0150] Table 6 Fluorine 18 F]Pyramide injection activity linear equation

[0151]

[0152] From Table 6 and Fig.11 It can be seen that: fluorine [ 18 The activity of [F]pyramide injection showed good linearity in the range of 2.8 ~ 797.5 μCi, with a correlation coefficient R of 0.9969.

[0153] Table 7 Fluorine 19 F] Pyramide content linear equation

[0154]

[0155] From Table 7 and Fig.12 It can be seen that: fluorine [ 18 Fluoride in pyramide injection 19 The content of [F]pyramide showed good linearity in the range of 0.22 ~33.30μg / mL, and the concentration level was 1% (LOQ) ~ 150%, with a correlation coefficient R of 1.0000.

[0156] 6. Accuracy

[0157] (1) Solution preparation

[0158] Blank solvent: 0.9% sodium chloride solution;

[0159] Content spike solution: prepared fluorine [ 19 F] The concentrations of pyramide reference solution were 0.44, 22.20, and 66.60 μg / mL;

[0160] Radiochemical purity spike solution: 20220704 batch labeled synthesis stock solution;

[0161] Test solution: 20240704 batch of fluorine [ 18 F] Pyramide injection;

[0162] Content accuracy test sample: Take fluorine [ 18 F] Pyramide injection was spiked with 0.44 μg / mL (1% spike level), 22.20 μg / mL (50% spike level), and 66.60 μg / mL (150% spike level) reference solution at a volume ratio of 1:1 to prepare accuracy spiked samples. Three samples were prepared at each concentration level, for a total of 9 samples.

[0163] Radiochemical purity accuracy test sample: Take the labeled synthetic mother solution as the impurity spiked sample and accurately determine its radioactivity concentration. Add the impurity sample to the injection product according to the volume ratio of the impurity sample to the injection solution of 10:90, 25:75, and 50:50, respectively, as the spiked test sample. Prepare 3 samples for each level at 3 spiked levels, for a total of 9 samples.

[0164] (2) Operation

[0165] fluorine[ 19 F] Accuracy of pyramide content: After the HPLC baseline is stable, according to the specified chromatographic conditions, inject no less than 1 injection of blank solution, 2 injections of test sample solution, 2 injections of labeled synthetic mother solution sample (radiochemical purity spiked solution), and 1 injection of each test sample for content accuracy, calculate the recovery rate, and evaluate the accuracy of the content method.

[0166]

[0167] in:

[0168]

[0169] As is the accuracy of the test sample fluorine [ 19 F] Pyramide peak area; A STD is the average peak area of ​​6 reference solutions; m STD is the weight of the reference solution (μg); Pw is the content of the reference; V STD Prepare volume (mL) for reference solution.

[0170] The matrix amount is the test sample fluorine [ 19 F] Pyramide content measurement value, calculated using the same formula as the measured amount, μg / mL;

[0171] The spiked amount is the weighted prepared content of the spiked reference substance, μg / mL.

[0172] Accuracy of radiochemical purity: After the baseline of the HPLC is stable, according to the specified chromatographic conditions, inject no less than 1 injection of blank solution, 2 injections of test sample solution, 2 injections of labeled synthesis mother liquor sample, and 9 portions of test sample, with 1 injection for each portion. Calculate the radiochemical purity and the recovery rate of total activity to evaluate the accuracy of radiochemical purity.

[0173]

[0174] Where: The measured value of radiochemical purity is the fluorine [ 18 F] pyramide percentage; the theoretical value of radiochemical purity is the fluorine [ 18 F] pyramide purity × test sample volume ratio + fluorine in mother liquor [ 18 F] Pyramide purity × spike ratio.

[0175] (3) Acceptable standards

[0176] fluorine[ 19F] The recovery rate of 1% spiked pyramide content should be 70~125%, and the relative standard deviation of the recovery rate within the group should be ≤20%; the recovery rate of 50% and 150% spiked levels should be 85~115%, and the relative standard deviation of the recovery rate within the group should be ≤5%.

[0177] The recoveries of the samples at all levels of radiochemical purity should be between 85% and 115%, and the relative standard deviation of the within-group recoveries should be ≤2%.

[0178] (4) Results

[0179] fluorine[ 18 Fluoride in pyramide injection 19 The test results of the spiked recovery rate of the [F]-pyramide content and the recovery rate of the radiochemical purity are shown in Tables 8 and 9, respectively.

[0180] Table 8 Fluorine 18 Fluoride in pyramide injection 19 F] Pyramide content spike recovery test results

[0181]

[0182] Table 9 Fluorine 18 Results of the radiochemical purity spike recovery test of [F]pyramide injection

[0183]

[0184] It can be seen from Tables 8 and 9 that: Fluorine [ 19 The recovery rate of 1% spiking level of pyramide content was 110.9%, and the relative standard deviation of the intra-group recovery rate was 0.13%; the recoveries of 50% and 150% spiking levels were 102.1% and 101.8%, respectively, and the relative standard deviations of the intra-group recoveries were 0.24% and 0.52%, respectively; the recoveries of the samples with different spiking levels of radiochemical purity were 102.8%~110.2%, and the relative standard deviation of the intra-group recoveries was ≤0.09%.

[0185] The above results show that this method can effectively 18 F]pyramide injection radiochemical purity and fluorine [ 19 F] The pyramide content was determined with good accuracy.

[0186] 7. Precision

[0187] 7.1 Repeatability

[0188] (1) Solution preparation

[0189] Blank solvent: 0.9% sodium chloride solution;

[0190] Test solution: 20240705 batch of fluorine [ 18 F] Pyramide injection;

[0191] Reference solution: 22.20 μg / mL fluoride 19 F] Pyramide reference solution;

[0192] Repeatability test solution: Take the test solution and the reference solution, mix them in equal proportions by volume, and prepare 6 parallel copies.

[0193] (2) Operation

[0194] After the HPLC baseline is stable, inject at least 1 injection of blank solution, 6 injections of reference solution, and 1 injection of each of 6 repeated test solutions, and record the radioactive chromatogram and UV chromatogram.

[0195] (3) Acceptable standards

[0196] The relative standard deviation of the radiochemical purity of 6 replicate test solution should be ≤2.0%;

[0197] In the UV spectrum of the test solution, fluorine [ 19 F] The relative deviation of the pyramide content value should be ≤2.0%.

[0198] (4) Results

[0199] Radiochemical purity and fluorine [ 19 The results of the repeatability test of the pyramide content are shown in Tables 10 and 11.

[0200] Table 10 Fluorine 18 Results of radiochemical purity and precision test of F]pyramide injection

[0201]

[0202] Table 11 Fluorine 18 Fluoride in pyramide injection 19 F] Pyramide content precision test results

[0203]

[0204] It can be seen from Tables 10 and 11 that the relative standard deviation of the radiochemical purity of the six replicate test solutions is 0.30%; 19 The relative deviation of the pyramide content value is 1.24%. The quality control method provided in this embodiment has good repeatability.

[0205] 7.2 Intermediate precision

[0206] (1) Solution preparation

[0207] Blank solvent: 0.9% sodium chloride solution;

[0208] Reference solution: 22.20 μg / mL fluoride 19 F] Pyramide reference solution;

[0209] Intermediate precision test solution: The second experimenter shall prepare 6 portions of intermediate precision test solution in parallel according to the preparation method of repeatability test solution.

[0210] (2) Operation

[0211] The intermediate precision of the test sample was tested using the same method as the repeatability on different HPLC instruments and chromatographic columns of the same type but different batches.

[0212] (3) Acceptable standards

[0213] The test results of the 6 intermediate precision samples should meet the acceptance criteria under the repeatability item;

[0214] The radioactive spectra of 12 test sample solutions were fluorine [ 18 The relative standard deviation of the radiochemical purity of F]pyramide should be ≤2.0%;

[0215] In the UV spectra of 12 test sample solutions, fluorine [ 19 F] The relative standard deviation of pyramide content should be ≤5.0%.

[0216] (4) Results

[0217] The intermediate precision test results are shown in Tables 10 and 11.

[0218] It can be seen from Tables 10 and 11 that the relative standard deviation of the radiochemical purity of the 6 intermediate precision test solutions is 0.13%; 19 The relative deviation of the pyramide content value was 0.62%; the relative standard deviation of the radiochemical purity of the 12 test solutions with the same repeatability was 0.38%; the relative standard deviation of the fluorine content of the 12 test solutions with the same repeatability was 0. 19 The relative standard deviation of the content of fluoro[ 18 F]pyramide radiochemical purity and fluorine [ 19 F] The pyramide content was detected with good precision.

[0219] 8. Durability

[0220] (1) Solution preparation

[0221] Blank solvent: 0.9% sodium chloride solution;

[0222] Sensitivity solution: Fluorine [ 19 F] A reference solution containing approximately 0.22 μg / mL of pyramide;

[0223] Durability test sample: Take the mother solution rich in impurities after the labeling reaction and mix it with the reference solution in equal volume ratio and shake well.

[0224] operate

[0225] Study the effect of method parameter changes on test results to evaluate method durability. According to the conditions in the table below, keep other parameters consistent with the specified parameters, change only one parameter at a time, and inject 1 injection of sensitivity solution and 2 injections of durability test sample in sequence.

[0226]

[0227] The mobile phase ratio is the weight ratio of anhydrous sodium dihydrogen phosphate to disodium hydrogen phosphate dihydrate required to prepare 1000 mL of mobile phase, and the phosphate concentration in the above mobile phase is 10 mmol / L.

[0228] (3) Acceptable standards

[0229] Under all parameter conditions, the signal-to-noise ratio of the sensitivity solution should be ≥10;

[0230] Fluorine[ 19 F]pyramide main peak and adjacent impurity peak separation should be ≥1.5; in the radioactive spectrum, fluorine [ 18 The separation degree between the main peak of [F]pyramide and the adjacent impurity peaks should be ≥1.5, and the relative standard deviation of radiochemical purity should be ≤5%.

[0231] If the set parameters do not meet the acceptance criteria, the parameter range can be narrowed and the experiment can be repeated.

[0232] (4) Results

[0233] fluorine[ 19 F] The durability test results of pyramide content are shown in Tables 12-1 and 12-2 (F-19 in the table represents fluorine [ 19 F]pyramide); the results of the radiochemical purity durability test are shown in Tables 13-1 and 13-2 (in the table, F-18 represents fluorine [ 18 F]pyramide).

[0234] Table 12-1 Fluorine 19 F] Pyramide content durability test results (20240718)

[0235]

[0236] Table 12-2 Fluorine 19F] Pyramide content durability test results (20240719)

[0237]

[0238] Table 13-1 Fluorine 18 F]Pyramide radiochemical purity durability test results (20240718)

[0239]

[0240] Table 13-2 Fluorine 18 F]Pyramide radiochemical purity durability test results (20240719)

[0241]

[0242] It can be seen from Tables 12-1, 12-2, 13-1 and 13-2 that under various parameter conditions, the fluorine content in the sensitivity solution [ 19 F]pyramide signal-to-noise ratio should be ≥10, which meets the requirements; compared with the initial conditions, the mobile phase composition changes, the flow rate ±0.2mL / min, and the column temperature ±5℃, the fluorine [ 19 F]pyramide main peak and adjacent impurity peak separation ≥ 2.1; in the radioactive spectrum, fluorine [ 18 The separation degree between the main peak of [F]pyramide and the adjacent impurity peaks is ≥1.6, and the relative standard deviation of radiochemical purity is ≤0.57%.

[0243] In the intermediate precision verification, chromatographic columns of the same model but different batches were used on different detection instruments. The relative standard deviation of the radiochemical purity of 12 test solutions according to the repeatability experiment results was 0.38%, proving that the method has good durability under the condition of replacing the chromatographic column.

[0244] The results of the durability experiments all met the acceptance criteria, and the method had good durability.

[0245] Through the above verification, the quality control method provided in this embodiment has good reliability. On this basis, blank solution, sensitivity solution, reference solution, verification reference solution, and test solution are taken and injected into the liquid chromatograph respectively to record the chromatogram. The UV spectrum of the blank solution is as follows Figure 1 The UV spectrum of the reference solution is as follows: Figure 2 As shown, check the UV spectrum of the reference solution as Figure 3 As shown, the UV and radioactivity spectra of the test solution are as follows Figure 4 and Figure 5 As shown, reasonable integration, in the UV spectrum according to the external standard method combined with its linear regression equation to calculate the fluorine [ 18 Fluoride in pyramide injection 19F]pyramide content, calculated by peak area normalization method in radioactive spectrum [ 18 F] Pyramide radiochemical purity.

[0246] from Figure 5 It can be seen that at 6.839min [ 18 F] pyramide peak; combined Figure 5 , it can be seen Figure 4 The 6.845min is fluorine [ 19 F] pyramide peak, and other impurity peaks all peaked within 8 minutes; therefore, using the quality control method of this embodiment, [ 18 F] Testing of pyramide injection.

[0247] After calculation, the results of multiple batches are: Fluorine [ 18 The radiochemical purity of pyramide was 95.0% to 99.8%, and the fluorine [ 19 F] The pyramide content is 0.2μg / mL~5.0μg / mL.

[0248] Comparative Example 1 (Mobile Phase Condition 1)

[0249] This comparative example provides a fluorine [ 18 F] The quality control method of pyramide injection is basically the same as that of Example 1, except that 0.1% phosphoric acid water is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed according to the following gradient. In this comparative example, the precursor compound is used as the analysis object.

[0250]

[0251] In this comparative example, the UV spectrum obtained is as follows Fig.13 As shown, it can be seen that the precursor compound is eluted in 2-3 minutes.

[0252] In Comparative Example 1, the precursor compound is used as the analysis object. According to the precursor compound and fluorine [ 19 F] pyramide structure can be judged that the polarity of the precursor compound is less than that of fluorine [ 19 F] pyramide polarity, therefore, when the mobile phase conditions of Comparative Examples 1-3 were applied to fluorine [ 18 F] pyramide injection, fluorine in the UV spectrum 19 F]pyramide peaks earlier, that is, the fluorine [ 19 F]pyramide will be eluted earlier, so the mobile phase conditions of this comparative example cannot achieve the fluorine [ 18 F] Separation of impurities and main components in pyramide injection.

[0253] Comparative Example 2 (Gradient Condition 1)

[0254] This comparative example provides a fluorine [ 18 F] The quality control method of pyramide injection is basically the same as that of Example 1, except that 10 mM phosphate at pH 7 is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed according to the following gradient.

[0255]

[0256] In this comparative example, the spectrum obtained is as follows Fig.14 and Fig.15 As shown, it can be seen that under the elution gradient of this comparative example, fluorine [ 18 F] pyridine peaked at 12.298 min, fluorine [ 19 F]pyridine peaked at 12.302 min, and Fig.15 It shows that the elution time of other impurities in the injection solution is even more than 30 minutes. These results indicate that the detection time is long when the gradient elution in this comparative example is used, the organic impurities are concentrated in the later peaks, and the separation degree between impurities does not meet the requirements.

[0257] Comparative Example 3 (Gradient Condition 2)

[0258] This comparative example provides a fluorine [ 18 F] The quality control method of pyramide injection is basically the same as that of Example 1, except that 10 mM phosphate at pH 7 is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed according to the following gradient.

[0259]

[0260] In this comparative example, the spectrum obtained is as follows Fig.16 and 17 As shown, it can be seen that under the elution gradient of this comparative example, fluorine [ 18 F] pyridine peaked at 9.906 min, fluorine [ 19 F]pyridine peaked at 9.903 min, and Fig.17 It shows that the elution time of other impurities in the injection is concentrated in 14-20 minutes, and even exceeds 20 minutes. These results indicate that when the gradient in this comparative example is used for elution, the main component still has a late elution time and a long detection time.

[0261] By comparing Example 1 with Comparative Examples 2 and 3, it can be seen that when the compositions of mobile phases A and B are the same, only due to the different elution gradients, the elution gradients in Comparative Examples 2 and 3 lead to a late peak time of the main component, and the peak time of the impurities (ultraviolet impurities) in the injection solution is concentrated and delayed, and the separation degree cannot meet the requirements, resulting in a prolonged detection time, thereby affecting the activity of the radioactive drug; the elution gradient in Example 1 can not only achieve effective separation of impurities and the main component, but also achieve efficient completion of fluorine [ 18 F] Testing of pyraclostrobin injection.

[0262] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorine 18 F] A quality control method for pyramide injection, characterized in that: Including the use of high performance liquid chromatography to detect the fluorine [ 18 F]pyramide radiochemical purity, fluoro[ 19 F] pyramide content, at least one of the precursor compounds, the high performance liquid chromatography method uses a chromatographic column with octadecylsilane bonded silica gel as a filler, a 10mM phosphate buffer with a pH of 6-8 as a mobile phase A, and acetonitrile as a mobile phase B, and elution is performed according to the following gradient: 0-7.5 min, 70-90% mobile phase A and 10-30% mobile phase B; 7.5-10 min, 60-80% mobile phase A and 20-40% mobile phase B; 10-14.9 min, 20-30% mobile phase A and 70-80% mobile phase B; 14.9-15min, mobile phase A increased from 20-30% to 70-90% and mobile phase B decreased from 70-80% to 10-30%; 15-20 min, 70-90% mobile phase A and 10-30% mobile phase B; Wherein, the fluorine [ 18 F] Pyramide has the following structure: ； The fluorine 19 F] Pyramide has the following structure: ； The precursor compound has the following structure: 。 2. The quality control method according to claim 1, characterized in that: The mobile phase A and mobile phase B are eluted according to the following gradient: 0-7.5 min, 75-85% mobile phase A and 15-25% mobile phase B; 7.5-10 min, 70-80% mobile phase A and 20-30% mobile phase B; 10-14.9 min, 15-25% mobile phase A and 75-85% mobile phase B; 14.9-15min, mobile phase A increased from 15-25% to 75-85% and mobile phase B decreased from 75-85% to 15-25%; 15-20 min, 75-85% mobile phase A and 15-25% mobile phase B.

3. The quality control method according to claim 1 or 2, characterized in that: The chromatographic column is C18, 4.6 mm×250 mm, 5 μm.

4. The quality control method according to claim 3, characterized in that: The column temperature of the chromatographic column is 25-35°C.

5. The quality control method according to claim 1, 2 or 4, characterized in that: The mobile phase A and the mobile phase B were eluted at a flow rate of 0.8-1.2 mL / min.

6. The quality control method according to claim 5, characterized in that: The mobile phase A and the mobile phase B were eluted at a flow rate of 1.0 mL / min.

7. The quality control method according to claim 1, 2, 4 or 6, characterized in that: The high performance liquid chromatography method uses an ultraviolet detector to detect fluorine [ 19 F] Pyramide and / or its content.

8. The quality control method according to claim 7, characterized in that: The detection wavelength of the ultraviolet detector is 244-264nm.

9. The quality control method according to claim 8, characterized in that: Also includes the use of radioactive flow detectors to detect fluorine [ 18 F] Radiochemical purity of pyramide.

10. The quality control method according to claim 1 or 2 or 4 or 6 or 8 or 9, characterized in that: The labeled synthetic stock solution is used as the impurity test sample and / or radiochemical purity spike solution.

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