Quality control method of fluorine [< 18 > F] piramide injection

Abstract

The invention provides a quality control method of fluorine [< 18 > F] piramide injection, which adopts a high performance liquid chromatography, adopts a C18 chromatographic column, takes 10mM phosphate buffer with pH of 6-8 as a mobile phase A, takes acetonitrile as a mobile phase B, and performs gradient elution. The quality control method provided by the invention can effectively separate fluorine [19F] piramide and other ultraviolet impurities in the injection and effectively separate fluorine [18F] piramide and other radiochemical impurities, and provides a basis for an accurate detection result; the radiochemical purity of fluorine [18F] pyramide and the content and impurities of fluorine [19F] pyramide are measured at the same time by combining an ultraviolet detector and a radioactive flow detector. Operation is carried out for 20 min, detection is completed, and rapid inspection and release of the fluorine [18F] piramide injection are achieved; the marked synthetic mother liquor is used as an impurity test sample and / or a radiochemical purity marked solution, so that all impurities in the injection can be detected and the detection accuracy is ensured.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and more particularly to a fluorine […]. 18 F] Quality control methods for pyramide injection. Background Technology

[0002] fluorine[ 18 F]pyramide injection is a positron emission tomography (PET) radiopharmaceutical primarily used clinically for the diagnosis of melanoma patients or suspected melanoma patients expressing melanin. Fluorine [ 18 [F]Pyramide is the active ingredient in injectable products. Its proportion of radioactive substances in the injectable product, i.e., its radiochemical purity, is a key quality indicator. Currently, the detection method for radiochemical purity of radiopharmaceuticals is still mainly based on radioactive thin-layer chromatography. However, thin-layer chromatography usually cannot separate and quantify structurally similar impurities (including radiochemical impurities and organic impurities).

[0003] fluorine[ 18 F]pyramide injection is a precursor compound reacted with radioactive fluorine in a synthesizer. 18 The fluorine [F] ion was prepared by nucleophilic substitution reaction to obtain fluorine [F]. 18 F]pyramide, the synthetic route is shown below.

[0004]

[0005] fluorine[ 18 F]Pyramide Synthetic Route Various radioactive and organic impurities are generated during the preparation of the injection solution. Most of these impurities can be removed through separation and purification methods, including fluorine. 19 F]pyramide is a fluorinated [ 18 The stable isotope of F]pyramide is also a factor affecting drug quality, and accurate determination of its content is a necessary quality control item. Summary of the Invention

[0006] To accurately measure fluorine [ 19 The content of pyramide and fluorine [F] 18 The radiochemical purity of pyramide [F], the primary objective of this invention is to provide a fluorine [ 18 The quality control method for F]pyramide injection has achieved the goal of fluorine [ 19 Effective separation of pyramide and other UV impurities, fluorine [ 18 Effective separation of F]pyramide and other radiochemical impurities, for fluorine [ 19 The content of pyramide and fluorine [F] 18 This provides a basis for the accurate and simultaneous testing of the radiochemical purity of F]pyramide.

[0007] In some embodiments of the present invention, the quality control method includes high-performance liquid chromatography (HPLC), wherein the HPLC uses a C18 (octadecylsilane-bonded silica gel) column as the packing material, a 10 mM phosphate buffer solution with a pH of 6-8 as mobile phase A, and acetonitrile as mobile phase B, and elutes according to the following gradient (wherein the percentages of mobile phase A and mobile phase B are both volume percentages): 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; 14.9-15 min, mobile phase A increases from 20-30% to 70-90% and mobile phase B decreases from 70-80% to 10-30%; 15-20 min, 70-90% mobile phase A and 10-30% mobile phase B.

[0008] The quality control method provided by this invention uses mobile phase A and mobile phase B, and elutes according to the gradient, to remove ultraviolet impurities and fluorine from the injection solution. 19 Effective separation of F]pyramide, while simultaneously eliminating radiochemical impurities and fluorine in the injection solution. 18 The effective separation of F]pyramide provides a basis for further detection and, in accordance with the relevant ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines, determines whether further control or reporting of impurities in the drug is required to ensure the safety and efficacy of the drug.

[0009] In some embodiments of the present invention, mobile phase A and mobile phase B are eluted in the following gradient (wherein the percentages of mobile phase A and mobile phase B are both volume percentages): 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; 14.9-15 min, 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.

[0010] By employing the specific mobile phase elution gradient described above, both UV impurities and fluorine can be eluted. 19 Effective separation of F]pyramide, while also removing radioactive impurities and fluorine from the injection solution. 18 Effective separation of F]pyramide can also achieve more efficient detection, completing the separation of fluorine [F]pyramide in a short time. 18 Testing of F]pyramide injection.

[0011] In some embodiments of the present invention, the chromatographic column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

[0012] In some embodiments of the present invention, the column temperature of the chromatographic column is 25-35°C.

[0013] In some embodiments of the present invention, mobile phase A and 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.

[0014] In some embodiments of the present invention, the liquid chromatography method employs an ultraviolet detector to detect fluorine [ 19 The content of F]pyramide and / or the preferred detection wavelength is 254 nm.

[0015] The second objective of this invention is to provide a quality control method that can simultaneously achieve fluorine [ 18 Fluorine in pyramide injection [F] 19 F]pyramide and / or its content and fluorine [ 18 The radiochemical purity of pyramide was determined, and the aforementioned quality control methods were further combined with a radiofrequency detector to detect fluorine […]. 18 The radiochemical purity of pyramide is achieved by combining high-performance liquid chromatography (UV detector) with a radiation flow detector, enabling simultaneous determination of fluorine in the injectable product using a single detection method. 18 The radiochemical purity of pyramide and the fluorine content in the injection solution [F] 19 F]Pyramide content.

[0016] The third objective of this invention is to ensure that fluorine [ 18 All impurities in the F]pyramide injection solution can be detected. Based on the aforementioned scheme, the labeled synthetic mother liquor is used as the impurity test sample and / or the radiochemical purity spiked solution to ensure the specificity and accuracy of the method.

[0017] This invention utilizes a combination of high-performance liquid chromatography (with an ultraviolet detector) and a radioactive flow detector (Radio-HPLC) to simultaneously determine the radiochemical purity and fluorine content of injectable products. 19 Simultaneous determination of pyramide content and all impurities; detection can be completed in 20 minutes, realizing the determination of fluorine […].18 The rapid testing and release of F]pyramide injection is to meet the requirement that radiopharmaceuticals undergo release testing before clinical use. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The fluorine provided by this invention 18 One of the ultraviolet chromatograms of the blank solution involved in the quality control method of F]pyramide injection.

[0020] Figure 2 The fluorine provided by this invention 18 The quality control method for F]pyramide injection involves one of the ultraviolet spectra of the reference solution.

[0021] Figure 3 The fluorine provided by this invention 18 The quality control method for F]pyramide injection involves verifying the ultraviolet spectrum of the reference solution.

[0022] Figure 4 The fluorine provided by this invention 18 The quality control methods for pyramide injection involve fluorine [ 18 One of the UV spectra of the F]pyramide test sample.

[0023] Figure 5 The fluorine provided by this invention 18 The quality control methods for pyramide injection involve fluorine [ 18 One of the radiometric spectra of the F]pyramide test sample.

[0024] Figure 6 The fluorine provided by this invention 18 The quality control method for F]pyramide injection involves one of the impurities tested in the ultraviolet spectrum of the sample.

[0025] Figure 7 The fluorine provided by this invention 18 The quality control method for F]pyramide injection involves one of the radiometric spectra of the impurity test sample.

[0026] Figure 8 The fluorine provided by this invention 18 The quality control method for F]pyramide injection involves one of the ultraviolet spectra of the test sample after 4 hours of high-temperature degradation.

[0027] Figure 9 The fluorine provided by this invention 18 The quality control method for F]pyramide injection involves one of the radiometric spectra of the test sample after 4 hours of high-temperature degradation.

[0028] Figure 10 The fluorine provided by this invention 18 The quality control methods for pyramide injection involve fluorine [ 19 One of the UV spectra of F]pyramide limit of quantitation solution.

[0029] Figure 11 The fluorine provided by this invention 18 The quality control methods for pyramide injection involve fluorine [ 18 One of the linear graphs of pyramide activity.

[0030] Figure 12 The fluorine provided by this invention 18 The quality control methods for pyramide injection involve fluorine [ 19 One of the linear graphs of pyramide content [F].

[0031] Figure 13 The fluorine provided by this invention 18 The quality control method for F]pyramide injection is one of the ultraviolet spectra involved in Comparative Example 1.

[0032] Figure 14 The fluorine provided by this invention 18 The quality control method for F]pyramide injection is one of the radiochromatograms involved in Comparative Example 2.

[0033] Figure 15 The fluorine provided by this invention 18 The quality control method for F]pyramide injection is one of the ultraviolet spectra involved in Comparative Example 2.

[0034] Figure 16 The fluorine provided by this invention 18 The quality control method for F]pyramide injection involves one of the radiochromatograms in Comparative Example 3.

[0035] Figure 17 The fluorine provided by this invention 18 The quality control method for F]pyramide injection is one of the ultraviolet spectra involved in Comparative Example 3. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] Chemical pharmaceutical preparations typically consist of an active ingredient and excipients. The detection of the content or purity of the active ingredient in the preparation usually prioritizes analytical methods for the active ingredient. Radioactive fluorine [ 18 F-labeled drugs are typically obtained from precursor compounds through processes such as chemical reaction, separation and purification, and mixing with excipients to obtain the final injectable product.

[0038] The active ingredient fluoride in the injection solution involved in the embodiments of this invention [ 18 F]pyramide was obtained by labeling the precursor compound with the following structural formula.

[0039]

[0040] In the labeling reaction, the p-toluenesulfonic acid group TsO- of the precursor compound leaves and is replaced by fluorine. 18 F] ion substitution generates the radioactive active ingredient fluorine in the formulation. 18 F]pyramide, and simultaneously, a small amount of stable fluorine introduced from the reaction reagents, equipment, and piping. 19 F ions react with some precursor compounds to form stable fluorine [ 19 F]pyramide. The precursor compound is labeled to generate fluorine […]. 18 The solution following the labeling reaction of pyramide injection is called the labeling synthesis mother liquor, which contains, except for fluorine […]. 18 F]pyramide is also rich in precursor compounds and fluorine. 19 F]pyramide and other impurities, such as fluorine in the compound. 18 Radiochemical impurities, organic process impurities, and degradation impurities of [F]. Among them, fluorine [ 19 The content of pyramide (F) needs to be controlled to prevent it from reacting with radioactive fluorine. 18 The competitive role of F]pyramide in targeting lesions.

[0041] Existing technologies utilize aqueous trifluoroacetic acid and trifluoroacetic acid acetonitrile solution to separate fluorine. 19 F]pyramide or fluorine [ 18 F]pyramide, but the inventors discovered that fluorine [ 18 F]pyramide and fluorine [ 19F]pyramide is unstable in acidic environments; therefore, using an aqueous solution of trifluoroacetic acid or acetonitrile as the mobile phase for elution will lead to the depletion of fluorinated [[]pyramide]. 18 F] Pyramide decomposition affects the accuracy of detection data. The main analyte is poorly retained on the C18 column. If the above mobile phase is used, the analysis time must be extended, which is not conducive to the detection of radiopharmaceuticals. Furthermore, the method does not examine the applicability and specificity of the method for the detection of potential impurities, and cannot guarantee the effective identification and separation of structurally similar impurities, such as the RRT 0.92 impurity in this method, the structure of which is shown below (F element is...). 18 F or 19 F corresponds to either a radiochemical impurity or an ultraviolet impurity in the formulation that has the chemical structure.

[0042]

[0043] From a chemical structural analysis perspective, the precursor compound and fluorine [ 19 F]pyramide or fluorine [ 18 F]pyramides do not differ much, so formulation analysis methods can be developed based on precursor analysis methods, and the detection of precursor compound residues in formulations can be achieved.

[0044] Therefore, the fluorine [provided by the present invention] 18 The development of a quality control method for F]pyramide injection is based on the detection method of precursor compounds, and further optimizes conditions such as initial mobile phase ratio, elution gradient, and run time. Simultaneously, it uses a reaction mother liquor rich in impurities (labeled synthesis mother liquor) for fluorination. 19 F]pyramide and fluorine [ 18 By screening the testing conditions for pyramide [F] and selecting representative optimized conditions, the quality control method provided by this invention is obtained, in order to achieve the control of fluorine [F]. 18 F]Pyramide injection radiochemical purity and fluorine [ 19 The F]pyramide content and the detection of various actual impurities in the labeled synthesis mother liquor are ensured to be detected, and the analysis time is minimized to a minimum of 20 minutes.

[0045] The quality control method provided by this invention involves connecting an ultraviolet detector and a radiation flow detector in series. During detection, the sample flows through a chromatographic column and is eluted with a mobile phase. The eluent first passes through the ultraviolet detector for fluorine […]. 19 The detection of pyramide and ultraviolet impurities was performed, and then the eluent was passed through a radiation flow detector for fluorine […]. 18 Detection of radiochemical purity and radiochemical impurities of F]pyramide.

[0046] The information on reagents, instruments, etc. used in the embodiments of this invention is shown in the table below.

[0047]

[0048] In this embodiment of the invention, unless otherwise specified, the solutions involved are prepared according to the following method: Blank solution: 0.9% sodium chloride solution; Test solution: Fluorine [ 18 F] Pyramide Injection; Reference solution (100% level): Accurately weigh approximately 20 mg of fluoride [ 19 F]pyramide reference standard, placed in a 100 mL volumetric flask, dissolved in acetonitrile to prepare fluorinated [ 19 F] Pyramide reference stock solution (stored at -20℃); aliquot 2.0 mL of the reference stock solution into a 20 mL volumetric flask, dilute to volume with 0.9% sodium chloride solution to obtain fluorine [ 19 F]pyramide reference solution, with a content of approximately 20 μg / mL.

[0049] Sensitivity solution: Accurately measure 1 mL of the reference solution and place it in a 100 mL volumetric flask. Dilute to volume with blank solution and shake well to obtain a concentration of approximately 0.2 μg / mL (1% level).

[0050] Mobile phase A: Accurately weigh 0.540 g of anhydrous sodium dihydrogen phosphate and 0.979 g of disodium hydrogen phosphate (dihydrate), dissolve in water and bring the volume to 1000 mL, then degas by sonication for 15 minutes to obtain the mobile phase A.

[0051] Mobile phase B: Measure an appropriate amount of acetonitrile and degas it by sonication for 15 minutes.

[0052] The volume of the above solutions can be adjusted according to the dosage while ensuring the target concentration remains unchanged.

[0053] Example 1 This embodiment provides a fluorine [ 18 The quality control methods for pyramide injection include simultaneous detection of fluoride [F]. 18 F]Pyramide radiochemical purity and its fluorine content [ 19 The content of F]pyramide was determined by a combination of high performance liquid chromatography and radiation flow detector.

[0054] The conditions for high-performance liquid chromatography (HPLC) are as follows: Chromatographic column: C18 column (4.6mm × 250mm, 5μm); Mobile phase A: 10 mM phosphate buffer, pH 7.0; Mobile phase B: Acetonitrile; Gradient elution procedure:

[0055] Flow rate: 1.0 mL / min; Column temperature: 30℃; Ultraviolet detector wavelength: 254nm; Injection volume: 10 μL; Running time: 20 minutes.

[0056] First, the reliability of the quality control method provided in this embodiment is verified.

[0057] 1. System Applicability Blank solution: 0.9% sodium chloride solution; Reference solution: 22.20 μg / mL fluorine [ 19 F] Pyramide reference solution; Verification of reference solution: 22.48 μg / mL fluorine [ 19 F] Pyramide reference solution; Test solution: Batch 20240704 fluorine [ 18 F]Pyramide Injection.

[0058] After the baseline of the high-performance liquid chromatography (HPLC) has stabilized, inject the following solutions in sequence: inject at least one blank solution (10 μL), inject the reference solution six times consecutively, inject the verification reference solution once, and inject the fluorine […]. 18 [F]Pyramide test solution was injected six times consecutively, and the ultraviolet and radioactive signals were recorded separately. The chromatograms were then saved. Figure 1-5 As shown.

[0059] from Figures 1-5 It can be concluded that the blank solution did not interfere with the main peak of the reference solution in the ultraviolet light; the relative standard deviation of the peak area of ​​the main peak of the reference solution for 6 consecutive injections was 0.04% (meeting ≤2%), and the relative standard deviation of the retention time was 0.11% (meeting ≤1%); the recovery rate of the reference solution was 100.5% (meeting 95~105%); in the radiometric spectrum, the relative standard deviation of the retention time of the main radiometric peak of the 6 injections of the test solution was 0.08% (meeting ≤1.0%), and the relative standard deviation of the radiochemical purity was 0.05% (meeting ≤1.0%).

[0060] The above results demonstrate that the quality control method provided in Example 1 is system-suitable and has good sample injection precision.

[0061] 2. Specificity assessment (1) Solution preparation Blank solution: 0.9% sodium chloride solution; Test solution: Batch 20240704 fluorine [ 18 F] Pyramide Injection; Reference solution: 22.20 μg / mL fluorine [ 19 F] Pyramide reference solution; Specific spiked sample: Mix the test solution and the reference solution in equal volume proportions to obtain the sample; High-temperature degradation sample: Take a specific spiked sample and place it in a 60℃ stability chamber for 4 hours to obtain the sample. Impurity test sample: Batch 20240704, fluorine [ 18 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, it can be determined that all potential impurities can be detected.

[0062] (2) Operation After the baseline of the high-performance liquid chromatography (HPLC) has stabilized, inject the blank solution at least once, the reference solution six times consecutively, the specific spiked sample once, the impurity test sample once, and the high-temperature degradation sample once. Record the UV and radioactive signals respectively, and save the chromatograms. The UV and radioactive spectra of the impurity test sample are shown below. Figure 6 and Figure 7 As shown, where, Figure 7 Fluorine was present at 6.835 min. 18 The peak of pyramide [F] was observed at 2.425 min, indicating the presence of free fluorine. 18 The peak of F] ion, and the other peaks are other radiochemical impurity peaks; Figure 6 Fluorine was present at 6.843 min. 19 The peak of pyramide (F) was observed at 13 min, indicating the presence of the precursor compound; other peaks were those of UV chemical impurities. The UV and radiometric spectra of the sample after 4 hours of high-temperature degradation are shown below. Figure 8 and Figure 9 As shown, fluorine [ 18 The peak of pyramide was observed at 6.831 min (F). Figure 8 Other peaks are radiochemical impurity peaks), fluorine [ 19 The peak of F]pyramide was observed at 6.839 min.

[0063] (3) Acceptable standards In the UV spectra of specific spiked samples, fluorine [ 19 The ratio of the retention time of pyramide to the average retention time of the main peak of the reference solution (6 injections) should be between 0.98 and 1.02; fluorine in the impurity test sample and high-temperature degradation sample... 18 The resolution between the pyramide peak and the adjacent impurity peak should be ≥1.5, and the fluorine [ 19 The separation between the F]pyramide peak and the adjacent impurity peak should be ≥1.5.

[0064] (4) Results: As shown in Table 1.

[0065] Table 1 Specificity Validation Results

[0066] Table 1 shows that: Fluorine [ 19 The ratio of the retention time of pyramide to the average retention time of the main peak in the reference solution (6 injections) was 1.00 (meeting the requirement of 0.98-1.02); fluorine in the impurity test sample and the high-temperature degradation sample... 18 The resolution between the pyramide peak and the adjacent impurity peak was greater than 1.5, and the fluorine [ 19 The separation 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 embodiment has been verified, the method specificity is good, and the analyte is not affected by the matrix components.

[0067] 3. Limit of Quantification (1) Solution preparation Blank solution: 0.9% sodium chloride solution; Test solution: Fluorine [ 18 F] Pyramide Injection; fluorine[ 19 F] Pyramide limit of quantitation solution: Sensitivity solution, prepared in 6 parallel portions by diluting with reference solution; Limit of Quantification (LOQ) Solution: For the lowest concentration of the injection solution (10 mCi / mL), the radioactivity concentration after 10 hours is 0.23 mCi / mL. If the injection volume is 10 μl and the minimum activity of the injection is 2.3 μCi, then the LQ activity should be lower than 2.3 μCi. [Fluoride...] 18 F] Pyramide injection (test solution): Calculate the radioactivity concentration at the time of injection based on the half-life and injection sequence, dilute with blank solution to the expected radioactivity concentration, and prepare 6 portions of activity limit of quantitation solution.

[0068] (2) Operation After the baseline of the high-performance liquid chromatography (HPLC) stabilizes, inject six aliquots of the activity limit of quantitation solution sequentially with one injection each, and save the radiometric chromatogram (omitted) for evaluating the activity limit of quantitation; then inject six consecutive injections of the sensitivity solution (i.e., fluorine […]). 19 F]Pyramide limit of quantitation solution), save the UV spectrum, such as Figure 10 As shown, it is used to evaluate fluorine [ 19 Limit of quantification for pyramide (F) Figure 10 In the middle, fluorine was present at 6.839 min. 19 F]pyramide peak, solvent peak around 18 min.

[0069] (3) Acceptable standards In the radiochromatogram, fluorine in the activity limit solution [ 18The signal-to-noise ratio (S / N) of the pyramide peak is ≥10. The injection activity is calculated based on decay. The relative standard deviation of the activity values ​​for 6 injections should be ≤20%. The limit of quantitation activity level should be reported. In the UV spectrum of the sensitive solution, fluorine [ 19 The signal-to-noise ratio (S / N) of the pyramide peak should be ≥10; the relative standard deviation of the peak area should be ≤20.0%, and the limit of quantitation concentration level should be reported.

[0070] (4) Results: As shown in Table 2.

[0071] Table 2 Fluorine [ 18 F]Pyramide activity and fluorine [ 19 F] Validation results of the limit of quantitation of pyramide content

[0072] Table 2 shows that the average activity obtained from the activity limit of quantitation solution measurement was 2.7 μCi, and the average signal-to-noise ratio was 246; fluorine [ 19 The limit of quantification for pyramide F was 0.22 μg / mL, and the average signal-to-noise ratio was 83.

[0073] 4. Detection limit (1) Solution preparation Blank solution: 0.9% sodium chloride solution; fluorine[ 19 F] Pyramide detection limit solution: Accurately measure 5 mL of sensitivity solution, dilute with blank solution to 10 mL, and this is the detection limit solution; (2) Operation After the baseline of the high-performance liquid chromatography (HPLC) instrument stabilized, three consecutive injections of the limit of detection solution were performed, and the ultraviolet spectra were saved (omitted) for evaluation of fluorine […]. 19 F] pyramide detection limit.

[0074] (3) Acceptable standards In the UV spectrum of the detection limit solution, fluorine [ 19 If the signal-to-noise ratio (S / N) of the F]pyramide peak is ≥3, report the detection limit concentration level.

[0075] (4) Results: As shown in Table 3.

[0076] Table 3 Fluorine [ 19 F]Pyramide detection limit test results

[0077] As can be seen from Table 3, the quality control method provided in this embodiment, fluorine [ 19 The detection limit for pyramide content is 0.11 μg / mL.

[0078] 5. Linear range (1) Solution preparation Blank solvent: 0.9% sodium chloride solution; Activity linearity test solution: Take fluorine [ 18 F]pyramide injection, diluted with blank solution, sample information is shown in Table 4.

[0079] Table 4 Activity-Linearity Test Sample Information (July 5, 2024)

[0080] fluorine[ 19 F] Pyramide linear test solution: Accurately weigh 22.48 mg of fluorine [ 19 [F] Pyramide reference standard was placed in a 100 mL volumetric flask, dissolved in acetonitrile, and diluted to volume to prepare the reference standard stock solution. Accurately measure the reference standard stock solution and dilute it with blank solution to prepare the linear test solution, as shown in Table 5.

[0081] Table 5 Fluorine [ 19 F] Pyramide linear test sample information

[0082] (2) Operation After the baseline of the high-performance liquid chromatograph (HPLC) stabilizes, inject at least one injection of the blank solution and one injection each of the radiochemical purity linearity test solution, according to the specified chromatographic conditions. Save the chromatogram (details omitted). Plot a standard curve with radioactivity as the x-axis and peak area as the y-axis, calculate the regression equation, correlation coefficient, and list the sum of squared residuals. The theoretical value of radioactivity is calculated from the calibrated activity after decay correction.

[0083] Inject at least one blank solution, fluoride [ 19 Inject one sample of each linear pyramide test solution. Plot a standard curve with peak area on the x-axis and injection concentration on the y-axis. Calculate the regression equation, correlation coefficient, and list the residual sum of squares.

[0084] (3) Acceptable standards List the linear equation; the linear correlation coefficient R ≥ 0.99; report the sum of squared residuals; Activity linear range: Report activity from the limit of quantitation to the highest linear activity; fluorine[ 19 F] Pyramide linear range: Report the concentration from the limit of quantitation to the highest linear concentration and concentration level.

[0085] (4) Results fluorine[ 18 The linear equation and linear graph for the activity of pyramide are shown in Table 6. Figure 11 As shown; Fluorine [ 19The linear equation and linear graph for pyramide content are shown in Table 7. Figure 12 As shown.

[0086] Table 6 Fluorine [ 18 [F] Pyramide Injection Activity Linear Equation

[0087] From Table 6 and Figure 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.

[0088] Table 7 Fluorine [ 19 [F] Linear equation for pyramide content

[0089] From Table 7 and Figure 12 It can be seen that: fluorine [ 18 F] Fluoride in pyramide injection 19 The pyramide concentration exhibited good linearity in the range of 0.22 to 33.30 μg / mL, with a concentration level of 1% (LOQ) to 150% and a correlation coefficient R of 1.0000.

[0090] 6. Accuracy (1) Solution preparation Blank solvent: 0.9% sodium chloride solution; Spiked solution: Fluorine [content] 19 The concentrations of the pyramide reference solutions were 0.44, 22.20, and 66.60 μg / mL, respectively. Radiochemical purity spiked solution: batch labeled synthesis mother liquor, 20220704; Test solution: Batch 20240704 fluorine [ 18 F] Pyramide Injection; Content accuracy test sample: fluorine [ 18 F] Pyramide injection was prepared by adding reference solutions at a volume ratio of 1:1:0.44 μg / mL (1% spiking level), 22.20 μg / mL (50% spiking level), and 66.60 μg / mL (150% spiking level) to prepare accuracy spiking samples. Three samples were prepared for each concentration level, for a total of nine test samples.

[0091] Radiochemical purity accuracy test samples: The labeled synthetic mother liquor was used as the impurity additive sample, and its radioactivity concentration was accurately determined. The impurity sample was added to the injection product at volume ratios of 10:90, 25:75, and 50:50, respectively, as the spiked test samples. Three spiked levels were prepared, with three test samples prepared for each level, for a total of nine test samples.

[0092] (2) Operation fluorine[ 19 F] Accuracy of pyramide content: After the baseline of the high performance liquid chromatograph is stable, inject no less than 1 blank solution, 2 test solution, and 2 labeled synthetic mother liquor samples (radiochemical purity spiked solution) according to the specified chromatographic conditions. Inject each test sample once, calculate the recovery rate, and evaluate the accuracy of the content method.

[0093]

[0094] in:

[0095] As represents the accuracy of the fluorine content in the test sample. 19 F] Pyramide peak area; A STD The average peak area of ​​the reference solution is 6 m. STD Weigh the reference solution (μg); Pw is the content of the reference solution; V STD Prepare the volume (mL) for the reference solution.

[0096] The matrix amount is the fluorine in the test sample. 19 F] The measured value of pyramide content is calculated using the same formula as the measured amount, in μg / mL; The spiking amount is the content prepared by weighing the spiked reference standard, in μg / mL.

[0097] Radiochemical purity accuracy: After the baseline of the high performance liquid chromatograph stabilizes, inject at least one blank solution, two test solution, and two labeled synthetic mother liquor samples according to the specified chromatographic conditions. For the radiochemical purity accuracy, inject one sample of each of the nine test samples. Calculate the recovery rate of radiochemical purity and total activity, and evaluate the accuracy of radiochemical purity.

[0098]

[0099] Among them: the radiochemical purity measurement value is the fluorine [ calculated by the peak area normalization method of the spiked sample]. 18 F]Pyramide percentage; theoretical value of radiochemical purity is the fluorine content in the sample. 18 F] Pyramide purity × test sample volume ratio + fluoride in mother liquor[ 18 F]Pyramide purity × Spike ratio.

[0100] (3) Acceptable standards fluorine[ 19 The recovery rate of pyramide spiked at the 1% level should be 70-125%, and the relative standard deviation of the recovery rate within the group should be ≤20%; the recovery rate of pyramide spiked at the 50% and 150% levels should be 85-115%, and the relative standard deviation of the recovery rate within the group should be ≤5%.

[0101] The recoveries of the test samples at all spiking levels of radiochemical purity should be between 85% and 115%, and the relative standard deviation of the recoveries within the group should be ≤2%.

[0102] (4) Results fluorine[ 18 F] Fluoride in pyramide injection 19 The results of the spiked recovery rate of F]pyramide content and the recovery rate of radiochemical purity are shown in Tables 8 and 9, respectively.

[0103] Table 8 Fluorine [ 18 F] Fluoride in pyramide injection 19 F]Recovery test results of pyramide content spiked recovery rate

[0104] Table 9 Fluorine [ 18 Results of Radiochemical Purity Spike Recovery Test of Pyramide Injection

[0105] As can be seen from Tables 8 and 9: Fluorine [ 19 The recovery rate of the pyramide spike at the 1% level was 110.9%, with a relative standard deviation of 0.13% within the group; the recoveries at the 50% and 150% levels were 102.1% and 101.8%, respectively, with relative standard deviations of 0.24% and 0.52% within the group, respectively; the recoveries of the test sample at each spiking level for radiochemical purity ranged from 102.8% to 110.2%, with relative standard deviations of ≤0.09% within the group.

[0106] The above results demonstrate that this method is effective for fluorine [ 18 F]Pyramide injection radiochemical purity and fluorine [ 19 The determination of F]pyramide content has good accuracy.

[0107] 7. Precision 7.1 Repeatability

[0108] (1) Solution preparation Blank solvent: 0.9% sodium chloride solution; Test solution: Batch 20240705 fluoride [ 18 F] Pyramide Injection; Reference solution: 22.20 μg / mL fluorine [ 19 F] Pyramide reference solution; Repeatable test solution: Take the test solution and the reference solution, mix them in equal volume ratio, and prepare 6 parallel portions.

[0109] (2) Operation After the baseline of the high performance liquid chromatograph stabilizes, inject at least one blank solution, inject six reference solutions, and inject one of each of the six repeatable test solutions. Record the radiochromatogram and ultraviolet chromatogram.

[0110] (3) Acceptable standards The relative standard deviation of the radiochemical purity of 6 repeatable test solutions should be ≤2.0%; In the UV spectrum of the test solution, fluorine [ 19 The relative deviation of the pyramide content value should be ≤2.0%.

[0111] (4) Results Radiochemical purity and fluorine [ 19 The repeatability test results of F]pyramide content are shown in Tables 10 and 11.

[0112] Table 10 Fluorine [ 18 Results of Radiochemical Purity and Precision Test of F]Pyramide Injection

[0113] Table 11 Fluorine [ 18 F] Fluoride in pyramide injection 19 F] Precision test results of pyramide content

[0114] Tables 10 and 11 show that the relative standard deviation of the radiochemical purity of the six repeatable test solutions was 0.30%; the fluorine content of the test samples... 19 The relative deviation of the pyramide content value was 1.24%. The quality control method provided in this embodiment has good repeatability.

[0115] 7.2 Intermediate Precision (1) Solution preparation Blank solvent: 0.9% sodium chloride solution; Reference solution: 22.20 μg / mL fluorine [ 19 F] Pyramide reference solution; Intermediate precision test solution: The second experimenter prepared 6 intermediate precision test solutions in parallel, referring to the preparation method of repeatability test solution.

[0116] (2) Operation Intermediate precision of the test sample was tested using the same repeatability method on different high performance liquid chromatographs and different batches of the same type of column.

[0117] (3) Acceptable standards The test results of the six intermediate precision test samples should meet the acceptable standard under the repeatability item; The radiometric spectra of 12 test sample solutions, including those for repeatability, showed the presence of fluorine. 18 The relative standard deviation of the radiochemical purity of pyramide (F) should be ≤2.0%; In the UV spectra of 12 test solutions for repeatability, fluorine [ 19 The relative standard deviation of the pyramide content should be ≤5.0%.

[0118] (4) Results The intermediate precision test results are shown in Tables 10 and 11.

[0119] Tables 10 and 11 show that the relative standard deviation of the radiochemical purity of the six intermediate precision test solutions was 0.13%; the fluorine content of the test samples... 19 The relative deviation of the pyramide content was 0.62%; the relative standard deviation of the radiochemical purity of the 12 repeatability test solutions was 0.38%; and the relative standard deviation of the fluorine content in the 12 repeatability test solutions was [missing information]. 19 The relative standard deviation of the pyramide content was 0.96%. These results demonstrate that the quality control method provided in this embodiment is effective for fluorinated [[...]]. 18 F] Pyramide radiochemical purity and fluorine content in injection [ 19 The determination of F]pyramide content has good precision.

[0120] 8. Durability (1) Solution preparation Blank solvent: 0.9% sodium chloride solution; Sensitivity solution: Fluorine [ 19 [F] A reference solution containing approximately 0.22 μg / mL of pyramide; Durability test sample: Take the mother liquor rich in impurities after the labeling reaction and mix it with the reference solution in equal volume ratio, and shake well to obtain the sample.

[0121] operate The effect of changes in method parameters on test results is investigated to assess method robustness. Under the conditions in the table below, while keeping other parameters consistent with the specified parameters, only one parameter is changed at a time, and one injection of sensitivity solution and two injections of robustness test sample are injected sequentially.

[0122]

[0123] 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. The phosphate concentration in the mobile phase is 10 mmol / L.

[0124] (3) Acceptable standards Under all parameter conditions, the signal-to-noise ratio of the sensitivity solution should be ≥10; Fluorine in UV spectra under various parameter conditions 19 The separation between the main peak of pyramide and the adjacent impurity peak should be ≥1.5; in the radiometric spectrum, fluorine [ 18 The separation between the main peak of F]pyramide and the adjacent impurity peak should be ≥1.5, and the relative standard deviation of radiochemical purity should be ≤5%.

[0125] If the set parameters do not meet the acceptable standards, the parameter range can be narrowed and the experiment repeated.

[0126] (4) Results fluorine[ 19 The durability test results for fluorinated pyramide content are shown in Tables 12-1 and 12-2 (in the table, F-19 represents fluorinated pyramide). 19 F]pyramide); the results of radiochemical purity durability tests are shown in Tables 13-1 and 13-2 (in the table, F-18 represents fluorine [ 18 [F]pyramide).

[0127] Table 12-1 Fluorine [ 19 F]Pyramide content durability test results (20240718)

[0128] Table 12-2 Fluorine [ 19 F]Pyramide content durability test results (20240719)

[0129] Table 13-1 Fluorine [ 18 F] Pyramide Radiochemical Purity Robustness Test Results (20240718)

[0130] Table 13-2 Fluorine [ 18 F]Pyramide Radiochemical Purity Robustness Test Results (20240719)

[0131] As can be seen from Tables 12-1, 12-2, 13-1, and 13-2: Under various parameter conditions, the sensitivity of fluorine in the solution […]. 19The signal-to-noise ratio of pyramide [F] should be ≥10, meeting the requirements; compared with the initial conditions, under changes in mobile phase composition, flow rate ±0.2 mL / min, and column temperature ±5℃, the fluorine [F] in the UV spectrum [F] should be ≥10, meeting the requirements; 19 The separation between the main peak of pyramide and adjacent impurity peaks is ≥2.1; in the radiometric spectrum, fluorine [ 18 The separation degree between the main peak of F]pyramide and the adjacent impurity peak is ≥1.6, and the relative standard deviation of radiochemical purity is ≤0.57%.

[0132] In the intermediate precision validation, the same model of chromatographic column but different batches were used on different detection instruments. The relative standard deviation of the radiochemical purity of 12 test solutions was 0.38% compared with the repeatability test results, which proved that the method has good robustness under the condition of changing chromatographic columns.

[0133] The durability test results all met the acceptance criteria, and the method demonstrated good durability.

[0134] Through the above verification, the quality control method provided in this embodiment has good reliability. Based on this, blank solution, sensitivity solution, reference solution, verification reference solution, and test solution were injected into the liquid chromatograph, and the chromatograms were recorded. The ultraviolet chromatogram of the blank solution is shown below. Figure 1 The UV spectrum of the reference solution is as follows: Figure 2 As shown, the UV spectrum of the reference solution was checked. Figure 3 As shown, the UV and radiometric spectra of the test solution are as follows: Figure 4 and Figure 5 As shown, by reasonable integration, fluoride […] is calculated in the ultraviolet spectrum using the external standard method combined with its linear regression equation. 18 F] Fluoride in pyramide injection 19 The pyramide content was calculated using the peak area normalization method in the radiometric spectrum. 18 F]Pyramide radiochemical purity.

[0135] from Figure 5 It can be seen that at 6.839 min, [ 18 The peak of F]pyramide; combined with Figure 5 It can be seen Figure 4 Fluorine was present at 6.845 min. 19 The peak of pyramide [F] eluted, and all other impurity peaks eluted within 8 minutes; therefore, using the quality control method of this embodiment, the [F] elution can be completed within 8 minutes. 18 Testing of F]pyramide injection.

[0136] Calculations showed that the results for multiple batches were: fluorine [ 18 The radiochemical purity of pyramide (F) is 95.0%~99.8%, and the fluorine [ 19The pyramide content ranges from 0.2 μg / mL to 5.0 μg / mL.

[0137] Comparative Example 1 (Mobile Phase Condition 1) This comparative example provides a fluorine [ 18 The quality control method for pyramide injection F] is basically the same as in 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 analyte.

[0138]

[0139] In this comparative example, the obtained ultraviolet spectra are as follows: Figure 13 As shown, the precursor compound was eluted in 2-3 minutes.

[0140] In Comparative Example 1, the precursor compound was used as the analytical object, based on the precursor compound and fluorine [ 19 The structure of F]pyramide indicates that the precursor compound is less polar than fluorine []. 19 The polarity of F]pyramide means that when the mobile phase conditions of Comparative Examples 1-3 are applied to fluorine [ 18 F]Pyramide injection, fluorine in UV spectrum [ 19 F]pyramide elutes earlier, that is, the fluorine [ 19 F]pyramide will be eluted earlier; therefore, using the mobile phase conditions in this comparative example, it is impossible to achieve fluorine [ 18 Separation of impurities from the main component in F]pyramide injection.

[0141] Comparative Example 2 (Gradient Condition 1) This comparative example provides a fluorine [ 18 The quality control method for F]pyramide injection is basically the same as that in Example 1, except that: 10mM 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.

[0142]

[0143] In this comparative example, the obtained spectra are as follows: Figure 14 and Figure 15 As shown, it can be seen that under the elution gradient of this comparative example, fluorine [ 18 F]pyridine eluted at 12.298 min, fluorine [ 19 F]pyridine eluted at 12.302 min, and Figure 15The results showed that the elution time of other impurities in the injection solution even exceeded 30 minutes. These results indicate that using the gradient elution method in this comparative example resulted in a long detection time, with organic impurities concentrated in the later elution stage, and the separation degree between impurities did not meet the requirements.

[0144] Comparative Example 3 (Gradient Condition 2) This comparative example provides a fluorine [ 18 The quality control method for F]pyramide injection is basically the same as that in Example 1, except that: 10mM 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.

[0145]

[0146] In this comparative example, the obtained spectra are as follows: Figure 16 and 17 As shown, it can be seen that under the elution gradient of this comparative example, fluorine [ 18 F]pyridine eluted at 9.906 min, fluorine [ 19 F]pyridine eluted at 9.903 min, and Figure 17 The results showed that the peak times of other impurities in the injection were concentrated between 14-20 min, or even more than 20 min. These results indicate that when using the gradient elution method in this comparative example, the main component still had a late peak time and a long detection time.

[0147] Comparing Example 1 with Comparative Examples 2 and 3, it can be seen that, with the same composition of mobile phases A and B, the elution gradients in Comparative Examples 2 and 3, due to their different gradients alone, result in a later peak time for the main component, and the peak times of impurities (UV impurities) in the injection solution are concentrated and delayed, and the separation degree cannot meet the requirements, leading to a prolonged detection time and thus affecting the activity of the radiopharmaceutical. The elution gradient in Example 1 effectively separates impurities from the main component and efficiently completes the separation of fluorine […]. 18 Testing of F]pyridine injection.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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: The method comprises adopting a high performance liquid chromatography method, wherein the high performance liquid chromatography method adopts a chromatographic column with octadecylsilane bonded silica gel as a filler, adopts a 10 mM phosphate buffer with a pH of 6-8 as a mobile phase A, adopts acetonitrile as a mobile phase B, and elutes 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.

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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