Thin layer chromatography detection method for free fluorine-18 in fluorine-18 labeled radiopharmaceutical

Abstract

The invention relates to the technical field of thin-layer chromatography detection, and discloses a thin-layer chromatography detection method for free fluorine-18 in a fluorine-18 labeled radiopharmaceutical, which adopts ethyl acetate and n-hexane as developing solvents, and the volume ratio of ethyl acetate to n-hexane is (50-90): (10-50). The developing solvent with a specific volume ratio is adopted, and the two key indexes that the Rf value of the marked active ingredient in the radiopharmaceutical is smaller than 0.8 and the separation degree (the separation degree of the marked active ingredient in the radiopharmaceutical and free fluorine-18) R is larger than 1 are considered.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of thin layer chromatography detection, in particular to a thin layer chromatography method for detecting free fluorine -18 in a fluorine -18 labeled radiopharmaceutical. BACKGROUND

[0002] Although there are thin layer chromatography methods for detecting free fluorine-18 in a fluorine -18 labeled radiopharmaceutical (for example fluorine [18F] ethyl-L-tyrosine injection) in the prior art, the labeled active ingredient in the radiopharmaceutical obtained by the method has a high Rf value (Rf≥0.8), and the two key indicators, i.e. Rf value <0.8 of the labeled active ingredient in the radiopharmaceutical and separation degree (separation degree of the labeled active ingredient and free fluorine 18 in the radiopharmaceutical) R>1, cannot be considered at the same time. When Rf value≥0.8, the active ingredient peak of the radiopharmaceutical appears outside the integral zone in the spectrum obtained by the thin layer scanner, thereby causing inaccurate or undetectable detection results. SUMMARY

[0003] Therefore, the present application provides a thin layer chromatography method for detecting free fluorine -18 in a fluorine -18 labeled radiopharmaceutical, so as to solve the problem that the two key indicators, i.e. Rf value <0.8 of the labeled active ingredient in the radiopharmaceutical and separation degree (separation degree of the labeled active ingredient and free fluorine -18 in the radiopharmaceutical) R>1, cannot be considered at the same time. Optionally, the thin layer chromatography method provided by the present application can reduce the development time. Optionally, the spectrum obtained by the thin layer chromatography method provided by the present application has a good peak shape. Optionally, the thin layer chromatography method provided by the present application achieves good recovery rate and / or RSD, and has good accuracy. Optionally, the thin layer chromatography method provided by the present application can consider the two key indicators, i.e. Rf value <0.8 of the labeled active ingredient in the radiopharmaceutical and separation degree (separation degree of the labeled active ingredient and free fluorine -18 in the radiopharmaceutical) R>1, at the same time when a specific volume ratio of developing agent and thin layer plate is used, and has good durability.

[0004] In a first aspect, the present application provides a thin layer chromatography method for detecting free fluorine -18 in a fluorine -18 labeled radiopharmaceutical, wherein the developing agent used is ethyl acetate and n-hexane, and the volume ratio of ethyl acetate and n-hexane is (50-90):(10-50). By using a specific volume ratio of developing agent, the thin layer chromatography method provided by the present application can consider the two key indicators, i.e. Rf value <0.8 of the labeled active ingredient in the radiopharmaceutical and separation degree (separation degree of the labeled active ingredient and free fluorine -18two key indicators (separation of the peaks of interest) R > 1.

[0005] In an alternative embodiment, the present application provides a method for the detection of free fluorine -18 In a labeled radiopharmaceutical by thin layer chromatography, the volume ratio of ethyl acetate and n-hexane is 60:40, 62:38 or 58:42. -18

[0006] In an alternative embodiment, the present application provides a method for the detection of free fluorine -18 In a labeled radiopharmaceutical by thin layer chromatography, the width of the thin layer plate is 1 cm and the length is 7-10 cm. -18

[0007] In an alternative embodiment, the present application provides a method for the detection of free fluorine -18 In a labeled radiopharmaceutical by thin layer chromatography, the thin layer plate is a silica gel plate, preferably DC-Fertigfolien -18

[0008] SIL G, TLC silica gel 60 F254 or POLYGRAM SIL G UV254.

[0009] In an alternative embodiment, the present application provides a method for the detection of free fluorine -18 In a labeled radiopharmaceutical by thin layer chromatography, the fluorine -18 labeled radiopharmaceutical is fluorine [18F] ethyl-L-tyrosine injection. -18

[0010] In an alternative embodiment, the present application provides a method for the detection of free fluorine -18 In a labeled radiopharmaceutical by thin layer chromatography, the volume of the sample is 1-5 μl. -18

[0011] In an alternative embodiment, the present application provides a method for the detection of free fluorine -18 In a labeled radiopharmaceutical by thin layer chromatography, the radioactivity distribution is determined by thin layer scanning of the thin layer plate after development with the developing agent. -18

[0012] In an alternative embodiment, the present application provides a method for the detection of free fluorine -18 In a labeled radiopharmaceutical by thin layer chromatography, the sample is diluted quantitatively to about 2-5 MBq / ml by methanol or water and / or spotted by capillary or pipette. -18 BRIEF DESCRIPTION OF DRAWINGS ​​​​​​​

[0013] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the accompanying drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the accompanying drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort based on these drawings.

[0014] Figure 1 is the chromatogram of Example 1 of the present application. A is the spectrum obtained by the thin layer scanner when the developing agent is methanol-acetic acid with a volume ratio of 90:10, B is the spectrum obtained by the thin layer scanner when the developing agent is methanol-water with a volume ratio of 90:10, C is the spectrum obtained by the thin layer scanner when the developing agent is acetonitrile-water with a volume ratio of 30:70, D is the spectrum obtained by the thin layer scanner when the developing agent is methanol-water with a volume ratio of 70:30, and E is the spectrum obtained by the thin layer scanner when the developing agent is methanol-water with a volume ratio of 50:50.

[0015] Figure 2 is the chromatogram of Example 1 of the present application. A is the spectrum obtained by the thin layer scanner when the developing agent is ethyl acetate-n-hexane with a volume ratio of 10:90, B is the spectrum obtained by the thin layer scanner when the developing agent is ethyl acetate-n-hexane with a volume ratio of 50:50, C is the spectrum obtained by the thin layer scanner when the developing agent is ethyl acetate-n-hexane with a volume ratio of 90:10, D is the spectrum obtained by the thin layer scanner when the developing agent is ethyl acetate-n-hexane with a volume ratio of 70:30, and E is the spectrum obtained by the thin layer scanner when the developing agent is ethyl acetate-n-hexane with a volume ratio of 60:40.

[0016] Figure 3 is the chromatogram of Example 2 of the present application. A is the spectrum obtained by the thin layer scanner when the solvent is methanol and the size of the thin layer plate is 1 cm x 7 cm, B is the spectrum obtained by the thin layer scanner when the solvent is water and the size of the thin layer plate is 1 cm x 7 cm, C is the spectrum obtained by the thin layer scanner when the solvent is methanol and the size of the thin layer plate is 1 cm x 10 cm, and D is the spectrum obtained by the thin layer scanner when the solvent is water and the size of the thin layer plate is 1 cm x 10 cm.

[0017] Figure 4 is the chromatogram of Example 3 of the present application. A is the spectrum obtained by the thin layer scanner when the sample volume is 1 μl, B is the spectrum obtained by the thin layer scanner when the sample volume is 2 μl, C is the spectrum obtained by the thin layer scanner when the sample volume is 3 μl, and D is the spectrum obtained by the thin layer scanner when the sample volume is 5 μl.

[0018] Figure 5is a chromatogram of Example 4 of the present application. A is a spectrum obtained by the thin layer scanner with capillary spotting, B is a spectrum obtained by the thin layer scanner with pipette spotting.

[0019] Figure 6 is a chromatogram of Example 5 of the present application. A is a spectrum obtained by the thin layer scanner with ethyl acetate-hexane (volume ratio 62:38) as developing agent, B is a spectrum obtained by the thin layer scanner with ethyl acetate-hexane (volume ratio 58:42) as developing agent, C is a spectrum obtained by the thin layer scanner with silica gel plate DC-Fertigfolien

[0020] SIL G as developing agent, D is a spectrum obtained by the thin layer scanner with silica gel plate POLYGRAM SIL G UV254.

[0021] In the diagram, contents means counts, position means position. DETAILED DESCRIPTION

[0022] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0023] The present application will be further described below in connection with specific embodiments, which should not be understood as limiting the scope of the present application.

[0024] EOS time is the end of synthesis time.

[0025] Investigation of developing agent in Example 1

[0026] (1) Method parameters:

[0027] Table 1

[0028] Method parameters Parameter content Thin layer plate TLC silica gel 60 F254 (brand Merck) Thin layer plate size 1 cm x 7 cm (developing distance 5 cm) Solvent Methanol Loading volume 5 μl Spotting method Pipette Instrument Thin layer chromatography scanner B-AR-2000-1 (brand Eckert & Ziegler) Instrument scanning time 60s

[0029] (2) Solution preparation

[0030] Test solution: take the test product (fluoro[18F]ethyl-L-tyrosine injection, the radioactivity concentration of the product is about 3700 MBq / ml at the EOS time) in an appropriate amount, dilute with methanol to about 2 MBq / ml (the loading volume is 5 μl, and the loading activity (radioactivity activity divided by volume is equal to radioactivity concentration) is about 10 KBq).

[0031] (3) Developing agent parameters:

[0032] Table 2

[0033] Type Volume ratio Methanol-acetic acid 90∶10 Methanol-water 90∶10 Acetonitrile-water 30∶70 Methanol-water 70∶30 Methanol-water 50∶50 Ethyl acetate-n-hexane 10∶90 Ethyl acetate-n-hexane 50∶50 Ethyl acetate-n-hexane 90∶10 Ethyl acetate-n-hexane 70∶30 Ethyl acetate-n-hexane 60∶40

[0034] (4) Assay method

[0035] The test sample solution was spotted on different thin layer plates, and developed in different developing solvents. After development, the radioactive distribution was determined by a thin layer scanner.

[0036] (5) Results and conclusions

[0037] Table 3 Methanol and acetonitrile developing solvents

[0038] Type Proportion Rf value Resolution R Methanol-acetic acid 90∶10 0.9 2.37 Methanol-water 90∶10 0.9 NA Acetonitrile-water 30∶70 0.9 2.20 Methanol-water 70∶30 0.9 NA Methanol-water 50∶50 0.8 2.00

[0039] NA indicates that the separation degree cannot be detected due to poor separation effect.

[0040] The results in Table 3 show that when the developing solvents are methanol-water and acetonitrile-water systems, the Rf value is greater than or equal to 0.8.

[0041] Table 4 Ethyl acetate-n-hexane system developing solvent

[0042] Type Proportion Rf value Resolution R Ethyl acetate-n-hexane 10∶90 0.02 NA Ethyl acetate-n-hexane 50∶50 0.5 1.12 Ethyl acetate-n-hexane 90∶10 0.7 1.57 Ethyl acetate-n-hexane 70∶30 0.6 1.55 Ethyl acetate-n-hexane 60∶40 0.5 1.43

[0043] NA indicates that the separation degree cannot be detected due to poor separation effect.

[0044] When the developing solvent is an ethyl acetate-n-hexane system with a volume ratio of (50-90):(10-50), the Rf value is less than 0.8.

[0045] By Figure 1 and Figure 2 It can be determined that when the Rf value is greater than or close to 0.8, the chromatographic peak of the labeled active ingredient in the radiopharmaceutical appears outside the integration zone, and the peak shape is not good. When the Rf value is less than 0.8, the active ingredient peak of the radiopharmaceutical appears in the integration zone, and the peak shape is good. The judgment standard of good peak shape is that there is a complete peak shape on the left side of the red vertical line in the figure.

[0046] Example 2 Investigation of solvents and thin layer plate sizes

[0047] (1) Method parameters:

[0048] Table 5

[0049]

[0050]

[0051] (2) Solution preparation

[0052] Test solution: Take the test sample (same as Example 1) by an appropriate amount, and dilute with methanol and water respectively to about 5 MBq / ml (the loading volume is 2 μl, and the loading activity is about 10 KBq).

[0053] (3) Thin layer plate specification and solvent parameters:

[0054] Table 6

[0055] Thin layer plate specification (width x length) Solvent type 1 cm x 7 cm (developing distance 5 cm) Methanol 1 cm x 7 cm (developing distance 5 cm) Water 1 cm x 10 cm (developing distance 8 cm) Methanol 1 cm x 10 cm (developing distance 8 cm) Water

[0056] (4) Determination method

[0057] Take the above test sample solution, and load it respectively on the above different thin layer plates, and develop it in the developing agent. After the development is completed, determine the radioactive distribution by a thin layer scanning instrument.

[0058] (5) Results and discussion

[0059] Table 7 Thin layer plate size, solvent type, Rf value and separation degree

[0060] Thin layer plate size Solvent type Rf value Resolution R 1 cm x 7 cm (developing distance 5 cm) Methanol 0.560 1.29 1 cm x 7 cm (developing distance 5 cm) Water 0.597 1.57 1 cm x 10 cm (developing distance 8 cm) Methanol 0.615 2.35 1 cm x 10 cm (developing distance 8 cm) Water 0.600 2.44

[0061] The Rf value (Rf value < 0.8) and the separation degree R (R > 1) of the two sizes of thin layer plates all meet the requirements, and the peak shape of both is good. However, the development time of the 1 cm x 10 cm size thin layer plate is longer, about 11 min, while the development time of the 1 cm x 7 cm size thin layer plate is about 3 min.

[0062] This example proves that even if the test sample is diluted with water or methanol, the radioactive drug can be separated and detected by the method of the present application, and the peak shape (refer to Figure 3 ) and the separation degree R all meet the requirements.

[0063] Example 3 Investigation of loading volume

[0064] The sample of this example is prepared as follows:

[0065] (1) Method parameters:

[0066] Table 8

[0067] Method parameters Parameter content Developing agent Ethyl acetate-n-hexane (60:40) Thin layer plate TLC silica gel 60 F254 (brand Merck) Thin layer plate size 1 cm x 7 cm (developing distance 5 cm) Solvent Methanol Spotting method Pipette Instrument Thin layer chromatography scanner B-AR-2000-1 (brand Eckert & Ziegler) Instrument scanning time 60s

[0068] (2) Solution preparation

[0069] Table 9 Loading concentration corresponding to different loading volumes

[0070] Loading volume Loading activity Loading concentration 1 μl 10 KBq 10 MBq / ml 2 μl 10 KBq 5 MBq / ml 3 μl 10 KBq 3.3 MBq / ml 5 μl 10 KBq 2 MBq / ml

[0071] According to Table 9, the test sample (same as Example 1) is diluted with methanol to the corresponding concentration.

[0072] (3) Assay method

[0073] The test sample solution was spotted on different thin layer plates and developed in different developing agents. After development, the radioactive distribution was determined by thin layer scanning.

[0074] (4) Results and discussion

[0075] It was found that the peak shape (reference Figure 4 ) and resolution R obtained met the requirements.

[0076] Investigation of spotting method in Example 4

[0077] (1) Method parameters:

[0078] Table 10

[0079] Method parameters Parameter content Developing agent Ethyl acetate-n-hexane (60:40) Thin layer plate TLC silica gel 60 F254 (brand Merck) Thin layer plate size 1 cm x 7 cm (developing distance 5 cm) Solvent Methanol Loading volume 2 μl Spotting method Capillary & pipette Instrument Thin layer chromatography scanner B-AR-2000-1 (brand Eckert & Ziegler) Instrument scanning time 60s

[0080] (2) Solution preparation

[0081] Test sample solution: an appropriate amount of test sample (the same as in Example 1) was diluted with methanol to about 5 MBq / ml (2 μl of sample volume, about 10 KBq of sample activity).

[0082] (3) Assay method

[0083] The diluted test sample solution was spotted on different thin layer plates by capillary and pipette, respectively, and developed in different developing agents. After development, the radioactive distribution was determined by thin layer scanning.

[0084] (4) Results and discussion

[0085] It was found that the peak shape (reference Figure 5 ) and resolution R obtained met the requirements.

[0086] Investigation of accuracy and robustness in Example 5

[0087] Accuracy

[0088] (1) Method parameters:

[0089] Table 11

[0090] Method parameters Parameter content Developing agent Ethyl acetate-n-hexane (60:40) Thin layer plate TLC silica gel 60 F254 (brand Merck) Thin layer plate size 1 cm x 7 cm (developing distance 5 cm) Solvent Methanol Loading volume 2 μl Spotting method Pipette Instrument Thin layer chromatography scanner B-AR-2000-1 (brand Eckert & Ziegler) Instrument scanning time 60s

[0091] (2) Recovery sample preparation

[0092] 1% spiked test sample (the same as in Example 1) solution preparation

[0093] The product and free fluorine -18The solution was diluted quantitatively to prepare a 1% spiked sample solution (5 KBq of the sample activity was loaded, and the product concentration was 2.5 MBq / ml, and the free fluorine concentration was 0.025 MBq / ml). One preparation was made, and three samples were loaded. 18 The solution was diluted quantitatively to prepare a 1% spiked sample solution (5 KBq of the sample activity was loaded, and the product concentration was 2.5 MBq / ml, and the free fluorine concentration was 0.025 MBq / ml). One preparation was made, and three samples were loaded.

[0094] 5% spiked sample solution preparation

[0095] The product and free fluorine were taken -18 The solution was diluted quantitatively to prepare a 5% spiked sample solution (10 KBq of the sample activity was loaded, and the product concentration was 5 MBq / ml, and the free fluorine concentration was 0.25 MBq / ml). One preparation was made, and three samples were loaded. -18 The solution was diluted quantitatively to prepare a 5% spiked sample solution (10 KBq of the sample activity was loaded, and the product concentration was 5 MBq / ml, and the free fluorine concentration was 0.25 MBq / ml). One preparation was made, and three samples were loaded.

[0096] 6% spiked sample solution preparation

[0097] The product and free fluorine were taken -18 The solution was diluted quantitatively to prepare a 6% spiked sample solution (20 KBq of the sample activity was loaded, and the product concentration was 10 MBq / ml, and the free fluorine concentration was 0.6 MBq / ml). One preparation was made, and three samples were loaded. -18 The solution was diluted quantitatively to prepare a 6% spiked sample solution (20 KBq of the sample activity was loaded, and the product concentration was 10 MBq / ml, and the free fluorine concentration was 0.6 MBq / ml). One preparation was made, and three samples were loaded.

[0098] (3) Determination method

[0099] The sample solution was taken and loaded on different thin layer plates with a pipette, and developed in a developing agent. After development, the radioactive distribution was determined by a thin layer scanner.

[0100] (4) Results and discussion

[0101] Table 12 Recovery rate results

[0102]

[0103] The background % means the free fluorine content in the unspiked sample, which was determined by a thin layer scanner.

[0104] The theoretical value % means the added amount of free fluorine.

[0105] The actual value % means the measured amount of free fluorine in the spiked sample.

[0106] Recovery rate (%) = (actual value - background) / added amount x 100%.

[0107] The recovery rate of each level was between 80% and 120%, and the RSD was less than 4% and less than 15%. The accuracy of the method was good.

[0108] Durability

[0109] (1) Method parameters:

[0110] Table 13

[0111] Method parameters Parameter content Developing agent Ethyl acetate - n-hexane (60:40) Thin layer plates TLC silica gel 60 F254 (brand Merck) Thin layer plate format 1 cm x 7 cm (developing distance 5 cm) Solvent Methanol Sample volume 2 μl Spotting method Pipette Instrument Thin layer chromatography scanner B-AR-2000-1 (brand Eckert & Ziegler) Instrument scan time 60s

[0112] (2) Robustness condition

[0113] Table 14

[0114]

[0115] (3) Test sample

[0116] The test sample was the 6% spiked test sample solution under the accuracy item above.

[0117] (4) Assay method

[0118] The test sample solution was taken and spotted on different thin layer plates with a pipette, and developed in different developing agents. After development, the radioactive distribution was determined by a thin layer scanner.

[0119] (5) Results and discussion

[0120] Table 15 Robustness test results

[0121]

[0122] Under the condition of ±2% fluctuation of the organic phase ratio, the Rf value (Rf value <0.8) and the resolution R (R>1) of the labeled active ingredient in the radiopharmaceutical could meet the requirements, and the peak shape was also good (refer to Figure 6 ); when two silica gel plates were used, the Rf value and the resolution R of the labeled active ingredient in the radiopharmaceutical could meet the requirements, and the peak shape was also good (refer to Figure 6 ). The method had good robustness.

[0123] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. All the embodiments do not need to be exhausted here. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A fluorine -18 A thin-layer chromatography method for the detection of free fluorine-18 in labeled radiopharmaceuticals, characterized in that, The developing solvent used is ethyl acetate and n-hexane, the volume ratio of ethyl acetate and n-hexane is (50-90):(10-50).

2. The fluorine according to claim 1 -18 Free fluorine in radiopharmaceuticals -18 detection method by thin layer chromatography, characterized in that, The volume ratio of ethyl acetate and n-hexane is 60:40, 62:38 or 58:

42.

3. Fluorine according to any one of claims 1-2 -18 Free fluorine in radiopharmaceuticals -18 A method for the detection of free fluorine in radiopharmaceuticals, characterized in that, The width of the thin layer plate is 1 cm, and the length is 7-10 cm.

4. Fluorine according to any one of claims 1 to 3 -18 Method for the detection of free fluorine in radiopharmaceuticals -18 characterized in that, The thin layer plate is a silica gel plate, preferably DC-Fertigfolien SIL G, TLC silica gel 60 F254 or POLYGRAM SIL G UV254.

5. Fluorine according to any one of claims 1 to 4 -18 Free fluorine in radiopharmaceuticals -18 A method for the detection of free fluorine in radiopharmaceuticals, characterized in that, The fluorine -18 The radiopharmaceutical is fluorine [18F] ethyl-L-tyrosine injection.

6. Fluorine according to any one of claims 1 to 5 -18 Free fluorine in radiopharmaceuticals -18 detection method by thin layer chromatography, characterized in that, The volume of spotting is 1-5 μl.

7. Fluorine according to any one of claims 1 to 6 -18 Free fluorine in radiopharmaceuticals -18 detection method by thin layer chromatography, characterized in that, The radioactivity distribution of the thin layer plate developed by the developing solvent is determined by a thin layer scanner.

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