TWI577990B - High performance liquid chromatography method for analyzing imaging agent, precursor of imaging agent, or intermediate of imaging agent - Google Patents

Description

High performance liquid chromatography method for contrast agent, contrast agent precursor or contrast agent intermediate

The present invention relates to an analytical method, and more particularly to a high performance liquid chromatography method suitable for contrast agents, contrast agent precursors or contrast agent intermediates.

Parkinson's disease is a neurodegenerative disease in which the pathogenesis is converted to the substantia nigra degeneration of the midbrain, causing the basal gangus to be degraded by the dopamine neurons of the caudate nucleus of the putamen, intersynaptic Dopamine levels decline and gradually lose mobility. The dopamine transporter is a special expression protein on dopamine neurons. As the neurons degenerate, the number of these dopamine transporter proteins is also reduced. The nuclear medicine scan uses a contrast agent that can label the dopamine transporter.鎝-99m TRODAT-1" can detect the number of dopamine neurons in the brain, and then evaluate whether the motor neurological disorder is caused by dopaminergic neurodegeneration, which can be used as an important basis for differential diagnosis.

For the process of the contrast agent TRODAT-1, there are many steps, and not every intermediate of the steps has a high performance liquid chromatography (HPLC) analysis method. A small number of existing analytical methods often fail to be rushed out during analysis due to low polarity impurities, often resulting in each analysis junction. There is a lack of reproducibility between fruits.

Conventional techniques use a single ratio of solvent as the extract, which can eliminate the dynamic phase equilibration time and rapid continuous injection analysis. However, when low-polarity impurities appear, the polarity of the mobile phase is often too low to be low. Polar impurities are detected from the column and are not detected. If the area percentage is used as the basis for judging the purity of the HPLC, when the presence of low-polarity impurities is not considered, the misjudgment of excessive purity is often caused. From another point of view, the low-polarity impurities that are not flushed out in the column may remain in the column, which may block the column or interfere with subsequent HPLC analysis, resulting in "hyperpurity" misjudgment. Therefore, it is necessary to develop new high performance liquid chromatography techniques to improve the above problems.

The main object of the present invention is to provide a high performance liquid chromatography method for analyzing a contrast agent, a contrast agent precursor or a contrast agent intermediate, which can be extracted by a high proportion of methanol to reduce the residue remaining in the column. The possibility of analyzing the column.

Another object of the present invention is to provide a high performance liquid chromatography method for contrast agent, contrast agent precursor or contrast agent intermediate, which can use UV of 210 nm as the detection wavelength in the analysis process, and can detect large Part of the molecule, with the correctness of the gain area percentage.

A further object of the present invention is to provide a high performance liquid chromatography method for contrast agent, contrast agent precursor or contrast agent intermediate, which introduces the concept of "gradient elution" to rush low-polar impurities with a high proportion of methanol. Out, and thus improve the credibility of the analysis results.

In order to achieve the above object, the present invention discloses a high performance liquid chromatography method for a contrast agent, a contrast agent precursor or a contrast agent intermediate, the contrast agent comprising a chemical structure: The method comprises the steps of: taking a contrast agent, a contrast agent precursor or a contrast agent intermediate on a chromatography column; using a first reduce agent, extracting the contrast agent, the contrast agent precursor or the a contrast agent intermediate, the first extracting agent comprises methanol and a concentration of 0.5 to 1% by weight of trifluoroacetic acid, and the volume ratio of the methanol to the first extracting agent is 5 to 70%; a flushing agent for extracting the contrast agent, the contrast agent precursor or the contrast agent intermediate, the second extracting agent comprising methanol and 0.5% to 1% by weight of trifluoroacetic acid, and the methanol accounts for The volume ratio of the second extracting agent is 95-99%; and the first flushing agent is used to dynamically extract the contrast agent, the contrast agent precursor or the contrast agent intermediate; The process uses a UV detector to record a chromatogram.

S1~S4‧‧‧ steps

1 is a flow chart showing the steps of an analysis method according to a preferred embodiment of the present invention; FIG. 2 is a chromatogram of a preferred embodiment of the present invention; FIG. 3 is a diagram showing A chromatogram of a preferred embodiment of the present invention; FIG. 4 is a chromatogram of a preferred embodiment of the present invention; and FIG. 5 is a layer of a preferred embodiment of the present invention Figure 6 is a chromatogram of a preferred embodiment of the present invention; Figure 7 is a chromatogram of a preferred embodiment of the present invention; Figure 8 is a chromatogram of a preferred embodiment of the present invention; Figure 9 is a chromatogram of a preferred embodiment of the present invention; Figure 10 is a A chromatogram of a preferred embodiment; and Figure 11 is a chromatogram of a preferred embodiment of the invention.

For a better understanding and understanding of the features and advantages of the present invention, the preferred embodiments and the detailed description are described as follows:

Referring to FIG. 1 , the present invention comprises the steps of high performance liquid chromatography for contrast agent, contrast agent precursor or contrast agent intermediate: step S1: taking a contrast agent, a contrast agent precursor or a contrast agent. The intermediate is on a chromatography column; Step S2: using a first reduce agent to extract the contrast agent, the precursor or the intermediate, the first reduce agent comprising methanol and a concentration by weight of 0.5~1 % trifluoroacetic acid, and the volume ratio of the methanol to the first extracting agent is 5 to 70%; Step S3: using a second extracting agent to extract the contrast agent, the precursor or the intermediate The second extracting agent comprises methanol and a concentration of 0.5 to 1% by weight of trifluoroacetic acid, and the proportion of the methanol to the second coating agent is 95 to 99%; and step S4: reusing the first A flushing agent is used to extract the contrast agent, the precursor or the intermediate.

Among them, in the process of moving, a UV detector is used to record a chromatogram.

In the process of flushing, the invention does not continuously use the flushing agent of the same composition, but firstly uses the first brewing agent with a lower proportion of methanol to move the extracted object, and then increases the proportion of methanol to 95-99. % as a second stripping agent for at least 15 minutes, and then flushed with the first stripping agent for at least 60 minutes to make a system for high performance liquid chromatography Stable back to the starting point. The reason for converting the first brewing agent to the second flushing agent is to detect the result of the contrast agent, the precursor or the intermediate by using UV to detect the first brewing agent. Further, it uses a UV having a detection wavelength of 210 nm, and after detecting the presence of the target substance, a signal is generated, and then the second flushing agent is used for the dynamic flushing.

The present invention uses 210 nm of UV as the detection wavelength system in order to make the percentage of the area of the UV absorption signal of the main component analyzed by the high performance liquid chromatography closer to the actual state of purity. Since benzene ring derivatives often have absorption at a wavelength of 254 nm, biochemical testers often use 254 nm as the UV detection wavelength. However, not all impurities have a benzene ring, that is, not all impurities are absorbed at 254 nm. Therefore, the present invention uses a UV wavelength of 210 nm as a detection wavelength to detect most molecules with a gain area. The correctness of the percentage.

Based on the foregoing steps, the present invention uses the contrast agent TRODAT-1 (its full name is 2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2. 1]-oct-2-yl]-methyl](2-mercaptoethyl)amino]ethyl]amino] ethanethiol-[1R-(extro-extro)]trihydrochloride, English 2- [[2-[[[3-(-Chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]-oct-2-yl]-methyl](2-mercaptoethyl)amino]ethyl]amino]ethanethiol- [1R-(exo-exo)]trihydrochloride, hereinafter abbreviated as TRODAT-1) is an operation example, and a Merck Chromolith RP-18e column is used as a chromatography column. The invention is applicable to the analysis of the final product, its precursor or its intermediate during the preparation of TRODAT-1.

In step S1, it is meant that the contrast agent, the contrast agent precursor or the intermediate in the contrast agent process has been produced according to a conventional process. In a preferred embodiment, the process of TRODAT-1 follows the synthetic route of Equations 1 and 2 below:

Taking the intermediate (1) in the formula 1 as an example, it is 2-(S-4-methoxybenzyl)thioethylamine, English is 2-(S-4-Methoxybenzyl)thioethylamine, and has the structure:

The analytical procedure was carried out in a 2.0 ml brown vial, and after weighing 4 mg of the intermediate (1), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 20% methanol/80% 0.1% aqueous trifluoroacetic acid as the first extract, wait 20 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 15 minutes. The first extract was replaced with 20% methanol/80% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 84 minutes. The retention time of the main signal (intermediate (1)) is approximately 12..63 minutes. Refer to the data and results in Table 1 and Figure 2 below.

Taking the precursor (2) in the formula 1 as an example, it is N-[2-(S-4-methoxybenzyl)thio]-2-chloroacetamide, which is N-[2- (S-4-Methoxybenzyl)thioethyl]-2-chloroacetamide, having the structure:

The analytical procedure was carried out in a 2.0 ml brown vial, and after weighing 4 mg of the precursor (2), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 50% methanol/50% 0.1% aqueous trifluoroacetic acid as the first extract, wait 20 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 15 minutes. The first extract was replaced with 50% methanol/50% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 84 minutes. The retention time of the main signal (precursor (2)) is approximately 7.41 minutes. Refer to the data and results in Table 2 and Figure 3 below.

Taking the intermediate (3) in the formula 2 as an example, it is a compound of (R)-(-)-citrastol methyl formate, and English is (R)-(-)-Anhydroecgonine methyl ester, having a structure of :

The analytical procedure was carried out in a 2.0 ml brown vial, and after weighing 4 mg of the intermediate (3), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 5% methanol/95% 0.1% aqueous trifluoroacetic acid as the first extract, wait 20 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 15 minutes. The first extract was replaced with 5% methanol/95% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 84 minutes. The retention time of the main signal (intermediate (3)) is approximately 6.13 minutes. Refer to the data and results in Table 3 and Figure 4 below.

Taking the intermediate (4) in the formula 2 as an example, it is a 2 β-methyl ester group 3 β-(4-chlorophenyl)decane, and English is 2 β-Carbomethoxy-3 β-(4-chlorophenyl). Tropane, with the structure:

The analytical procedure was carried out in a 2.0 ml brown vial, and after weighing 2.7 mg of the intermediate (4), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 40% methanol/60% 0.1% aqueous trifluoroacetic acid as the first extract, wait 15 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 40 minutes. The first extract was then changed to 40% methanol/60% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 64 minutes. The retention time of the main signal (intermediate (4)) is approximately 6.63 minutes. Refer to the data and results in Table 4 and Figure 5 below.

Taking the intermediate (5) in the formula 2 as an example, it is 2 β-carboxy-3 β-(4-chlorophenyl)decane, and English is 2 β-Carboxy-3 β-(4-chlorophenyl)tropane. , with a structure of:

The analytical procedure was performed in a 2.0 ml brown vial, and after weighing 4 mg of the intermediate (5), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 30% methanol/70% 0.1% aqueous trifluoroacetic acid as the first extract, wait for 30 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. Second flushing of aqueous acetic acid solution Liquid, then wait 40 minutes. The first extract was replaced with 30% methanol/70% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 64 minutes. The retention time of the main signal (intermediate (5)) is approximately 9.29 minutes. Refer to the data and results in Table 5 and Figure 6 below.

Taking the intermediate (6) in the formula 2 as an example, it is 3-(4-chlorophenyl)-N-[2-[S-(4-methoxybenzyl)thio]-ethyl] -8-azabicyclo[3.2.1]-octane-2-carboxamide-[1R-(extro-extro)), English is 3-(4-Chlorophenyl)-N-[2-[S-(4) -methoxybenzyl)thio]-ethyl]-8-azabicyclo[3.2.1]-octane-2-carboxamide-[1R-(exo-exo)], having the structure:

The analytical procedure was performed in a 2.0 ml brown vial and weighed 4 mg of intermediate. After (6), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 60% methanol/40% 0.1% aqueous trifluoroacetic acid as the first extract, wait 15 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 40 minutes. The first extract was replaced with 60% methanol/40% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 64 minutes. The retention time of the main signal (intermediate (6)) is about 6.30 minutes. Refer to the data and results in Table 6 and Figure 7 below.

Taking the intermediate (7) in the formula 2 as an example, it is 3-(4-chlorophenyl)-2-[[N-[2-[S-(4-methoxybenzyl)thio]] Ethyl]amino]methyl]-8-azabicyclo[3.2.1]octane [1R-(extro-extro)], English 3-(4-Chlorophenyl)-2-[[N-[2 -[S-(4-methoxybenzyl)thio]-ethyl]amino]methyl]-8-aza-bicyclo[3.2.1]octane-[1R-(exo-exo)], having the structure:

The analytical procedure was carried out in a 2.0 ml brown vial, and after weighing 3.3 mg of the intermediate (7), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 50% methanol/50% 0.1% aqueous trifluoroacetic acid as the first extract, wait 15 minutes, then change to 30% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 40 minutes. The first extract was replaced with 50% methanol/50% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 64 minutes. The retention time of the main signal (intermediate (7)) is approximately 6.19 minutes. Refer to the data and results in Table 7 and Figure 8 below.

Taking the intermediate (8) in the formula 2 as an example, it is 2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]-oct-2 -yl]methyl][2-[S-(4-methoxybenzyl)thio]ethyl]amino]-N-[2-[S-(4-methoxybenzyl)thio ]ethyl]acetamide-[1R-(extro-extroversion)], English is 2-[[[3-(4-Chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]-oct-2- Yl]methyl][2-[S-(4-methoxybenzyl)thio]ethyl]amino]-N-[2-[S-(4-methoxybenzyl)thio]ethyl]acetamide-[1R-(exo-exo)] , with a structure of:

The analytical procedure was performed in a 2.0 ml brown vial, and after weighing 4.5 mg of the intermediate (8), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 70% methanol/30% 0.1% aqueous trifluoroacetic acid as the first extract, wait for 15 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 40 minutes. The first extract was replaced with 70% methanol/30% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 64 minutes. The retention time of the main signal (intermediate (8)) is approximately 5.02 minutes. Refer to the data and results in Table 8 and Figure 9 below.

(Table 8)

Taking the intermediate (9) in the formula 2 as an example, it is 2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabi-ring [3.2.1] Oct-2-yl]methyl][[S-(4-methoxybenzyl)thio]ethyl]amino]ethyl]amino]-S-(4-methoxybenzyl) ethane sulfide Alcohol-[1R-(extro-extroversion)], English is 2-[[2-[[[3-(4-Chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]-oct-2-yl] Methyl][[S-(4-methoxybenzyl)thio]ethyl]amino]ethyl]amino]-S-(4-methoxybenzyl)ethanethiol-[1R-(exo-exo)], having the structure:

The analytical procedure was carried out in a 2.0 ml brown vial, and after weighing 4 mg of the intermediate (9), 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 60% methanol/40% 0.1% aqueous trifluoroacetic acid as the first extract, wait 30 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 15 minutes. Then change it to 60% methanol/40% 0.1% in 30 seconds. The first extract of the aqueous solution of fluoroacetic acid was finally waited for 74 minutes. The retention time of the main signal (intermediate (9)) is approximately 5.33 minutes. Refer to the data and results in Table 9 and Figure 10 below.

Taking the contrast agent TRODAT-1 of the formula 2 as an example, the analysis operation was carried out in a 2.0 ml brown sample vial, and after weighing 4 mg of the contrast agent TRODAT-1, 1.8 ml of methanol (HPLC grade) was added. The initial analysis conditions were as follows: 45% methanol/55% 0.1% aqueous trifluoroacetic acid as the first extract, wait 15 minutes, and then change to 99% methanol/1% 0.1% trifluorohydrate in 30 seconds. The second extract of the aqueous acetic acid solution was then waited for 20 minutes. The first extract was replaced with 60% methanol/40% 0.1% aqueous trifluoroacetic acid solution for 30 seconds, and finally waited for 84 minutes. The retention time of the main signal (contrast agent TRODAT-1) is about 4.15 minutes. Refer to the data and results in Table 10 and Figure 11 below.

For the analysis of each of the above examples, before the analysis of each sample, the ratio of the initial methanol to the aqueous solution of trifluoroacetic acid was further analyzed, and the phase was equilibrated for more than two hours, and then at least three times were analyzed with HPLC-grade methanol as a blank sample; After three blank test comparisons, it is confirmed that there is no residual component signal from the previous analysis, and then the sample is formally analyzed, so the score can be ensured. The correctness of the analysis.

The method for high performance liquid chromatography analysis of the contrast agent, the contrast agent precursor and the contrast agent intermediate disclosed in the invention can be extracted by a high proportion of methanol, and mixed with different ratios of methanol and trifluoroacetic acid. , reducing the possibility that the material that is easily retained in the tube remains in the analytical column; and allows the contrast agent, its precursor or its intermediate to be stably retained in the column, and obtain a reproducible main signal stagnation time; The invention further introduces the concept of "gradient flushing", which rushes out low-polarity impurities with a high proportion of methanol, thereby improving the credibility of the analysis results. Therefore, the present invention does have practical and economic value, and provides analytical techniques that have not been seen in the past.

The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and the variations, modifications, and modifications of the shapes, structures, features, and spirits described in the claims of the present invention. All should be included in the scope of the patent application of the present invention.

S1~S4‧‧‧ steps

Claims (6)

A high performance liquid chromatography method for contrast agent, contrast agent precursor or contrast agent intermediate, the contrast medium comprising a chemical structure: The method comprises the steps of: taking a contrast agent, a contrast agent precursor or a contrast agent intermediate on a chromatography column; using a first reduce agent, extracting the contrast agent, the contrast agent precursor or the a contrast agent intermediate, the first extracting agent comprises methanol and a concentration of 0.5 to 1% by weight of trifluoroacetic acid, and the volume ratio of the methanol to the first extracting agent is 5 to 70%; a flushing agent for extracting the contrast agent, the contrast agent precursor or the contrast agent intermediate, the second extracting agent comprising methanol and 0.5% to 1% by weight of trifluoroacetic acid, and the methanol accounts for The volume ratio of the second extracting agent is 95-99%; and the first flushing agent is used to dynamically extract the contrast agent, the contrast agent precursor or the contrast agent intermediate; The process uses a UV detector to record a chromatogram. A method for high performance liquid chromatography analysis of a contrast agent, a contrast agent precursor or a contrast agent intermediate according to claim 1, wherein the contrast agent and the precursor are dynamically extracted using the first extracting agent. Or in the step of the intermediate, the moving time is at least 20 minutes. A method for high performance liquid chromatography analysis of a contrast agent, a contrast agent precursor or a contrast agent intermediate according to claim 1, wherein the contrast agent and the precursor are dynamically extracted using the second extracting agent. Or in the step of the intermediate, the priming time is at least 15 minutes. A method for analyzing a contrast agent, a contrast agent precursor or a contrast agent intermediate according to claim 1, wherein the first agent is used to extract the contrast agent and the precursor In the step of the substance or the intermediate, the pulsing time is at least 60 minutes. A method for high performance liquid chromatography analysis of a contrast agent, a contrast agent precursor or a contrast agent intermediate according to claim 1, wherein the UV detector has a detection wavelength of 210 nm. A method for high performance liquid chromatography analysis of a contrast agent, a contrast agent precursor or a contrast agent intermediate according to claim 1, wherein the contrast agent precursor or the contrast agent intermediate comprises a group consisting of the following substances One of the groups: 2-(S-4-methoxybenzyl)thioethylamine, N-[2-(S-4-methoxybenzyl)thio]-2-chloroacetamide, (R)-(-)-Zhushui belladonic acid methyl ester, 2 β-methyl ester 3 β-(4-chlorophenyl)decane, 2 β-carboxy-3 β-(4-chlorophenyl ) decane, 3-(4-chlorophenyl)-N-[2-[S-(4-methoxybenzyl)thio]-ethyl]-8-azabicyclo[3.2.1]- Octane-2-carboxamide-[1R-(extro-extro)], 3-(4-chlorophenyl)-2-[[N-[2-[S-(4-methoxybenzyl)sulfide) Ethyl]amino]amino]methyl]-8-azabicyclo[3.2.1]octane [1R-(extro-extro)], 2-[[[3-(4-chlorophenyl)-8 -methyl-8-azabicyclo[3.2.1]-oct-2-yl]methyl][2-[S-(4-methoxybenzyl)thio]ethyl]amino]-N -[2-[S-(4-methoxybenzyl)thio]ethyl]acetamide-[1R-(extra-extroversion)] and 2-[[2-[[[3-(4- Chlorophenyl)-8-methyl-8-azabi-ring [3.2.1] xin -2-yl]methyl][[S-(4-methoxybenzyl)thio]ethyl]amino]ethyl]amino]-S-(4-methoxybenzyl)ethanethiol -[1R-(outward-extrovert)].