CN106770757A - A kind of method of separating and assaying of 9 demethyl α dihydrotetrabenazineins and its impurity - Google Patents
A kind of method of separating and assaying of 9 demethyl α dihydrotetrabenazineins and its impurity Download PDFInfo
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Abstract
The invention belongs to Pharmaceutical Analysis field, and in particular to the method for separating and assaying of a kind of 9 demethyl α dihydrotetrabenazineins and its impurity.The method of separating and assaying, 9 DM α DTBZ and its impurity α DTBZ and N methylaniline can be not only set to realize being kept completely separate, and the content of 9 DM α DTBZ and its impurity α DTBZ and N methylanilines can be simultaneously measured, for the assay of 9 demethyl α dihydrotetrabenazineins in 9 demethyl α dihydrotetrabenazinein bulk drugs and the assay about material provide a kind of easy, quick, sensitive detection method, so as to realize carrying out quality control to 9 DM α DTBZ bulk drugs, and then ensure PET imaging medicaments11The purity and mark rate of C α DTBZ, are conducive to PET imaging medicaments11Validity and security that C α DTBZ are used.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, and in particular to a kind of 9- demethyls-α-dihydrotetrabenazinein and its impurity
Method of separating and assaying.
Background technology
II type vesicular monoamine transporter (VMAT2) is a kind of memebrane protein on neuron intracytoplasmic pouch vacuolar membrane, in list
Key effect is played during the reuptake of amine mediator.Molecule clone technology identification show, VMAT2 be distributed mainly on the mankind and
In the central nervous system and pancreatic tissues of mammal.The change of VMAT2 is relevant with various diseases, such as Parkinson's (PD),
Alzheimer disease (AD), Huntington disease (HD), diabetes etc..In recent years, shown as target carries out radioactive tracer with VMAT2
Picture, relevant disease is early diagnosed, is classified, antidiastole, curative effect monitoring research, the focus as nuclear medicine studies it
One.Up to the present, the VMAT2 developers of clinic have been successfully applied to both at home and abroad, are contributed to positron emission emission computer and are broken
The positive electron tracer drug of layer (PET) imaging, such as：11C-α-DTBZ、18F-FP- α-DTBZ etc..
9- demethyls-α-dihydrotetrabenazinein (9-DM- α-DTBZ) is to prepare PET imaging medicaments11The mark of C- α-DTBZ
Precursor compound, labeling process is as follows：
Due to11C half-life period is only 20min, clinically uses tracer drug11C- α-DTBZ are needed by labelled precursor
Compound 9-DM- α-DTBZ are prepared in situ and obtain.The content of impurity has influence on PET and shows in labelled precursor compound 9-DM- α-DTBZ
As medicine11The purity and mark rate of C- α-DTBZ, further influence final imaging results.
At present, the 9-DM- α-DTBZ that have reported conventional preparation method is：α-dihydrotetrabenazinein (α-DTBZ) is in hydrogenation
Removed in the presence of sodium, HMPA and methylphenylamine methyl generation 9-DM- α-DTBZ (Chirality, 1997,
9：59-62).Experiment shows, after the completion of reaction, solvent and reaction raw materials sodium hydride, HMPA in reaction solution exist
Can be removed during post-reaction treatment, and reaction raw materials α-DTBZ and methylphenylamine then more difficult removal is clean.Therefore, exist
In the preparation process of 9-DM- α-DTBZ, the major impurity that may be mixed with is α-DTBZ and methylphenylamine.
Up to the present, the separation determination also not on 9-DM- α-DTBZ and its impurity α-DTBZ and methylphenylamine
The relevant report of method.In spite of document report：HPLC methods determine tetrabenazine bulk drug content, however, on the one hand due to 9-
The difference of DM- α-DTBZ bulk drugs impurities (predominantly α-DTBZ and methylphenylamine) and tetrabenazine bulk drug impurities
Not, on the other hand due to 9-DM- α-DTBZ and the difference of tetrabenazine chemical constitution, applicant is had found by studying, above-mentioned
HPLC methods determine tetrabenazine bulk drug content can not be used for the separation determination of 9-DM- α-DTBZ and its impurity, using this
During HPLC methods 9-DM- α-DTBZ be not completely separated with impurity, separating degree it is poor, and then cause 9-DM- α-DTBZ and its impurity
The content of α-DTBZ and methylphenylamine cannot be measured, and also just cannot carry out matter to the purity of 9-DM- α-DTBZ and content
Amount control, it is difficult to ensure PET imaging medicaments11The purity and mark rate of C- α-DTBZ, so as to be unfavorable for PET imaging medicaments11C-α-
Validity and security that DTBZ is used.
Therefore, set up the method for separating and assaying of a kind of 9- demethyls-α-dihydrotetrabenazinein and its impurity have it is important
Meaning.
The content of the invention
The invention solves the problems that first technical problem be that prior art cannot realize 9- demethyls-α-dihydrotetrabenazinein
The problem that 9-DM- α-DTBZ and its major impurity α-DTBZ and methylphenylamine in bulk drug are kept completely separate, the present invention will be solved
Second technical problem certainly be 9-DM- α during prior art cannot realize 9- demethyls-α-dihydrotetrabenazinein bulk drug-
The problem that the content of DTBZ and its major impurity α-DTBZ and methylphenylamine is measured simultaneously.
In order to solve the above technical problems, the present invention propose a kind of 9- demethyls-α-dihydrotetrabenazinein and its impurity α-
The method of separating and assaying of DTBZ and methylphenylamine.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions：
The present invention provides the method for separating and assaying of a kind of 9- demethyls-α-dihydrotetrabenazinein and its impurity, including following
Step：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 40~90 to add volume ratio：10~60
Ammonium acetate buffer-methyl alcohol is dissolved, and the solution that concentration is 0.5~1.5mg/mL is made, as need testing solution；
It is 40~90 with volume ratio with reverse-phase chromatographic column as chromatographic column according to high performance liquid chromatography：10~60 ammonium acetate
Buffer solution-methyl alcohol is mobile phase, and flow velocity is 0.5~1.5mL/min, and Detection wavelength is 270~290nm, and column temperature is 20~35 DEG C；
The μ L of the need testing solution 5~15 are taken, high performance liquid chromatograph is injected, determined.
Preferably, the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity, with volume
Than being 50~75：25~50 ammonium acetate buffer-methyl alcohol is mobile phase.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
It is 70 with volume ratio：30 ammonium acetate buffer-methyl alcohol is mobile phase；Or
It is 50 with volume ratio：50 ammonium acetate buffer-methyl alcohol is mobile phase；Or
It is 75 with volume ratio：25 ammonium acetate buffer-methyl alcohol is mobile phase.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
The concentration of the ammonium acetate buffer is 40~60mM, pH value is 4~5.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
The concentration of the ammonium acetate buffer is 50mM, pH value is 4.5.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
The filler of the reverse-phase chromatographic column is octadecylsilane chemically bonded silica.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
The filler of the reverse-phase chromatographic column is the octadecylsilane chemically bonded silica of 5 μm of particle diameter.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
The reverse-phase chromatographic column is 5 μm, the Sunfire C18 reverse-phase chromatographic columns of 4.6 × 150mm.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
Comprise the following steps：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 70 to add volume ratio：30 ammonium acetate delays
Fliud flushing-methyl alcohol is dissolved, and the solution that concentration is 1mg/mL is made, as need testing solution；
According to high performance liquid chromatography, the Sunfire C18 reverse-phase chromatographic columns with 5 μm, 4.6 × 150mm are chromatographic column, with body
Product is than being 70：30 ammonium acetate buffer-methyl alcohol is mobile phase, and flow velocity is 1mL/min, and Detection wavelength is 280nm, and column temperature is 25
℃；The concentration of the ammonium acetate buffer is 50mM, pH value is 4.5；
The μ L of the need testing solution 10 are taken, high performance liquid chromatograph is injected, determined；Or
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 50 to add volume ratio：50 ammonium acetate delays
Fliud flushing-methyl alcohol is dissolved, and the solution that concentration is 1.0mg/mL is made, as need testing solution；
According to high performance liquid chromatography, the Sunfire C18 reverse-phase chromatographic columns with 5 μm, 4.6 × 150mm are chromatographic column, with body
Product is than being 50：50 ammonium acetate buffer-methyl alcohol is mobile phase, and flow velocity is 1mL/min, and Detection wavelength is 280nm, and column temperature is 25
℃；The concentration of the ammonium acetate buffer is 50mM, pH value is 4.5；
The μ L of the need testing solution 10 are taken, high performance liquid chromatograph is injected, determined；Or
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 75 to add volume ratio：25 ammonium acetate delays
Fliud flushing-methyl alcohol is dissolved, and the solution that concentration is 1mg/mL is made, as need testing solution；
According to high performance liquid chromatography, the Sunfire C18 reverse-phase chromatographic columns with 5 μm, 4.6 × 150mm are chromatographic column, with body
Product is than being 75：25 ammonium acetate buffer-methyl alcohol is mobile phase, and flow velocity is 1mL/min, and Detection wavelength is 280nm, and column temperature is 25
℃；The concentration of the ammonium acetate buffer is 50mM, pH value is 4.5；
The μ L of the need testing solution 10 are taken, high performance liquid chromatograph is injected, determined.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
Also include following 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein reference substance solution, N- methylbenzenes
The step of preparation of amine reference substance solution and measure：
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 0.5~1.5mg/mL,
As 9- demethyls-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, the solution that concentration is 0.5~1.5mg/mL is made, as α-dihydro
Tetrabenazine reference substance solution；
The reference substance for taking methylphenylamine is appropriate, the solution that concentration is 0.5~1.5mg/mL is made, as methylphenylamine
Reference substance solution；
9- demethyls-α-dihydrotetrabenazinein the reference substance solution, the α-dihydrotetrabenazinein reference substance are taken respectively
Solution and each 5~15 μ L of the methylphenylamine reference substance solution, inject high performance liquid chromatograph, determine.
It is further preferred that the method for separating and assaying of above-mentioned 9- demethyls-α-dihydrotetrabenazinein of the invention and its impurity,
Also include following 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein reference substance solution, N- methylbenzenes
The step of preparation of amine reference substance solution and measure：
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, is gone as 9-
Methyl-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, as α-dihydro butylbenzene that
Piperazine reference substance solution；
The reference substance for taking methylphenylamine is appropriate, the solution that concentration is 1mg/mL is made, as methylphenylamine reference substance
Solution；
9- demethyls-α-dihydrotetrabenazinein the reference substance solution, the α-dihydrotetrabenazinein reference substance are taken respectively
Solution and each 10 μ L of the methylphenylamine reference substance solution, inject high performance liquid chromatograph, determine.
Compared with prior art, above-mentioned technical proposal of the invention has advantages below：
The method of separating and assaying of 9- demethyls-α-dihydrotetrabenazinein of the present invention and its impurity, with 5 μm, 4.6 × 150mm
Sunfire C18 reverse-phase chromatographic columns be chromatographic column, be 50~75 with volume ratio：25~50 ammonium acetate buffer-methyl alcohol is
Mobile phase, can not only make 9-DM- α-DTBZ and its impurity α-DTBZ and N- in 9- demethyls-α-dihydrotetrabenazinein bulk drug
Methylaniline is realized being kept completely separate, and can be same to the content of 9-DM- α-DTBZ and its impurity α-DTBZ and methylphenylamine
When be measured, be the assay of 9- demethyls-α-dihydrotetrabenazinein in 9- demethyls-α-dihydrotetrabenazinein bulk drug
Provide a kind of easy, quick, sensitive detection method with the assay about material such that it is able to realize to 9-DM- α-
DTBZ bulk drugs carry out quality control, and then ensure PET imaging medicaments11The purity and mark rate of C- α-DTBZ, are conducive to PET to show
As medicine11Validity and security that C- α-DTBZ are used.
Brief description of the drawings
In order that present disclosure is more likely to be clearly understood, below according to specific embodiment of the invention and combine
Accompanying drawing, the present invention is further detailed explanation, wherein：
Fig. 1 is the HPLC chromatogram of need testing solution in embodiment 1；
Fig. 2 is the HPLC chromatogram of 9- demethyls-α-dihydrotetrabenazinein reference substance in embodiment 1；
Fig. 3 is the HPLC chromatogram of α-dihydrotetrabenazinein reference substance in embodiment 1；
Fig. 4 is the HPLC chromatogram of methylphenylamine reference substance in embodiment 1；
Fig. 5 is the HPLC chromatogram of need testing solution in embodiment 2；
Fig. 6 is the HPLC chromatogram of need testing solution in embodiment 3；
Fig. 7 is the HPLC chromatogram of need testing solution in comparative example 1；
Fig. 8 is the HPLC chromatogram of need testing solution in comparative example 2；
Fig. 9 is the HPLC chromatogram of need testing solution in comparative example 3；
In Fig. 1-9, what 9-DM- α-DTBZ were represented is 9- demethyls-α-dihydrotetrabenazinein, and that α-DTBZ are represented is α-two
Hydrogen tetrabenazine.
Specific embodiment
In following examples of the present invention and experimental example, 9-DM- α-DTBZ represent for 9- demethyls-α-dihydro butylbenzene that
Piperazine, what α-DTBZ were represented is α-dihydrotetrabenazinein.
Embodiment 1
The method of separating and assaying of the present embodiment 9- demethyls-α-dihydrotetrabenazinein and its impurity, comprises the following steps：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 70 to add volume ratio：30 ammonium acetate delays
Fliud flushing (concentration is 50mM, pH value is 4.5)-methyl alcohol is dissolved, and is made the solution that concentration is 1mg/mL, molten as test sample
Liquid；
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, is gone as 9-
Methyl-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, as α-dihydro butylbenzene that
Piperazine reference substance solution；
The reference substance for taking methylphenylamine is appropriate, the solution that concentration is 1mg/mL is made, as methylphenylamine reference substance
Solution；
According to high performance liquid chromatography, with Sunfire C18 reverse-phase chromatographic columns (5 μm, 4.6 × 150mm) as chromatographic column, with body
Product is than being 70：30 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-methyl alcohol is mobile phase, and flow velocity is 1mL/min, inspection
Survey wavelength is 280nm, and column temperature is 25 DEG C；
Need testing solution, 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein control are taken respectively
Product solution and each 10 μ L of methylphenylamine reference substance solution, inject high performance liquid chromatograph, determine.
Need testing solution, 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein in the present embodiment
The HPLC chromatogram difference of reference substance solution and methylphenylamine reference substance solution is as Figure 1-4.
From Fig. 1-4, in embodiment 1, when 9-DM- α-DTBZ are with reaction raw materials α-DTBZ, the reservation of methylphenylamine
Between difference it is big, three is kept completely separate, good separation；The retention time of 9-DM- α-DTBZ is 6.22min, and content is
65.1%；The retention time of α-DTBZ is 10.60min, and content is 16.6%；The retention time of methylphenylamine is
13.88min, content is 18.3%.
Embodiment 2
The method of separating and assaying of the present embodiment 9- demethyls-α-dihydrotetrabenazinein and its impurity, comprises the following steps：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 50 to add volume ratio：50 ammonium acetate delays
Fliud flushing (concentration is 50mM, pH value is 4.5)-methyl alcohol is dissolved, and is made the solution that concentration is 1.0mg/mL, molten as test sample
Liquid；
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, is gone as 9-
Methyl-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, as α-dihydro butylbenzene that
Piperazine reference substance solution；
The reference substance for taking methylphenylamine is appropriate, the solution that concentration is 1mg/mL is made, as methylphenylamine reference substance
Solution；
According to high performance liquid chromatography, with Sunfire C18 reverse-phase chromatographic columns (5 μm, 4.6 × 150mm) as chromatographic column, with body
Product is than being 50：50 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-methyl alcohol is mobile phase, and flow velocity is 1mL/min, inspection
Survey wavelength is 280nm, and column temperature is 25 DEG C；
Need testing solution, 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein control are taken respectively
Product solution and each 10 μ L of methylphenylamine reference substance solution, inject high performance liquid chromatograph, determine.
The HPLC chromatogram of need testing solution is as shown in Figure 5 respectively in the present embodiment.
As shown in Figure 5, in embodiment 2,9-DM- α-DTBZ are slightly worse with reaction raw materials α-DTBZ separating effects；9-DM-α-
The retention time of DTBZ is 2.45min, and content is 65.4%；The retention time of α-DTBZ is 3.02min, and content is 15.4%；
The retention time of methylphenylamine is 6.68min, and content is 19.2%.
Embodiment 3
The method of separating and assaying of the present embodiment 9- demethyls-α-dihydrotetrabenazinein and its impurity, comprises the following steps：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 75 to add volume ratio：25 ammonium acetate delays
Fliud flushing (concentration is 50mM, pH value is 4.5)-methyl alcohol is dissolved, and is made the solution that concentration is 1mg/mL, molten as test sample
Liquid；
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, is gone as 9-
Methyl-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, as α-dihydro butylbenzene that
Piperazine reference substance solution；
The reference substance for taking methylphenylamine is appropriate, the solution that concentration is 1mg/mL is made, as methylphenylamine reference substance
Solution；
According to high performance liquid chromatography, with Sunfire C18 reverse-phase chromatographic columns (5 μm, 4.6 × 150mm) as chromatographic column, with body
Product is than being 75：25 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-methyl alcohol is mobile phase, and flow velocity is 1mL/min, inspection
Survey wavelength is 280nm, and column temperature is 25 DEG C；
Need testing solution, 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein control are taken respectively
Product solution and each 10 μ L of methylphenylamine reference substance solution, inject high performance liquid chromatograph, determine.
The HPLC chromatogram of need testing solution is as shown in Figure 6 respectively in the present embodiment.
It will be appreciated from fig. 6 that in embodiment 3,9-DM- α-DTBZ and reaction raw materials α-DTBZ, the retention time of methylphenylamine
Differ greatly, three is kept completely separate, separating effect is preferable；The retention time of 9-DM- α-DTBZ is 9.65min, and content is
66.2%；The retention time of α-DTBZ is 16.28min, and content is 16.7%；The retention time of methylphenylamine is
18.79min, content is 17.1%.
Embodiment 4
The method of separating and assaying of the present embodiment 9- demethyls-α-dihydrotetrabenazinein and its impurity, comprises the following steps：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 90 to add volume ratio：10 ammonium acetate delays
Fliud flushing (60mM, pH value are 4)-methyl alcohol is dissolved, and the solution that concentration is 1.5mg/mL is made, as need testing solution；
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, is gone as 9-
Methyl-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, as α-dihydro butylbenzene that
Piperazine reference substance solution；
The reference substance for taking methylphenylamine is appropriate, the solution that concentration is 1mg/mL is made, as methylphenylamine reference substance
Solution；
According to high performance liquid chromatography, with Sunfire C18 reverse-phase chromatographic columns (5 μm, 4.6 × 150mm) as chromatographic column, with body
Product is than being 90：10 ammonium acetate buffer (60mM, pH value are 4)-methyl alcohol is mobile phase, and flow velocity is 1.5mL/min, Detection wavelength
It is 270nm, column temperature is 35 DEG C；
Need testing solution, 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein control are taken respectively
Product solution and each 5 μ L of methylphenylamine reference substance solution, inject high performance liquid chromatograph, determine.
Embodiment 5
The method of separating and assaying of the present embodiment 9- demethyls-α-dihydrotetrabenazinein and its impurity, comprises the following steps：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 40 to add volume ratio：60 ammonium acetate delays
Fliud flushing (40mM, pH value are 5)-methyl alcohol is dissolved, and the solution that concentration is 0.5mg/mL is made, as need testing solution；
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, is gone as 9-
Methyl-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, as α-dihydro butylbenzene that
Piperazine reference substance solution；
The reference substance for taking methylphenylamine is appropriate, the solution that concentration is 1mg/mL is made, as methylphenylamine reference substance
Solution；
According to high performance liquid chromatography, with Sunfire C18 reverse-phase chromatographic columns (5 μm, 4.6 × 150mm) as chromatographic column, with body
Product is than being 40：60 ammonium acetate buffer (40mM, pH value are 5)-methyl alcohol is mobile phase, and flow velocity is 0.5mL/min, Detection wavelength
It is 290nm, column temperature is 20 DEG C；
Need testing solution, 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein control are taken respectively
Product solution and each 15 μ L of methylphenylamine reference substance solution, inject high performance liquid chromatograph, determine.
Embodiment 6
The present embodiment is differed only in embodiment 1：Flow velocity is 0.8mL/min；Remaining experiment condition and experimental implementation
Step is same as Example 1.
Embodiment 7
The present embodiment is differed only in embodiment 1：Flow velocity is 1.2mL/min；Remaining experiment condition and experimental implementation
Step is same as Example 1.
Comparative example 1
This comparative example is differed only in embodiment 1：In the step of preparing need testing solution, it is 20 to add volume ratio：
80 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-acetonitrile is dissolved；It is with volume ratio as need testing solution
20：80 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-acetonitrile is mobile phase；It is anti-phase with Lichrospher C18
Chromatographic column (5 μm, 4.6 × 250mm) is chromatographic column；Flow velocity is 0.8mL/min；Remaining experiment condition and laboratory operating procedures with
Embodiment 1 is identical.
The HPLC chromatogram of need testing solution is as shown in Figure 7 respectively in this comparative example.
As shown in Figure 7, in comparative example 1,9-DM- α-DTBZ are basically identical with the retention time of reaction raw materials α-DTBZ, peak
Shape almost overlaps, inferior separating effect；The retention time of 9-DM- α-DTBZ is 2.10min, and the retention time of α-DTBZ is
2.19min, the retention time of methylphenylamine is 3.00min.
Comparative example 2
This comparative example is differed only in comparative example 1：In the step of preparing need testing solution, it is 50 to add volume ratio：
50 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-acetonitrile is dissolved；It is with volume ratio as need testing solution
50：50 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-acetonitrile is mobile phase；Remaining experiment condition and experimental implementation
Step is identical with comparative example 1.
The HPLC chromatogram of need testing solution is as shown in Figure 8 respectively in this comparative example.
As shown in Figure 8, in comparative example 2, the separating degree of 9-DM- α-DTBZ and reaction raw materials α-DTBZ is 0.7, separating effect
Difference；The retention time of 9-DM- α-DTBZ is 2.24min, and the retention time of α-DTBZ is 1.73min, the reservation of methylphenylamine
Time is 6.93min.
Comparative example 3
This comparative example is differed only in comparative example 1：In the step of preparing need testing solution, it is 80 to add volume ratio：
20 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-acetonitrile is dissolved；It is with volume ratio as need testing solution
80：20 ammonium acetate buffer (concentration is 50mM, pH value is 4.5)-acetonitrile is mobile phase；Remaining experiment condition and experimental implementation
Step is identical with comparative example 1.
The HPLC chromatogram of need testing solution is as shown in Figure 9 respectively in this comparative example.
As shown in Figure 9, in comparative example 3, together with 9-DM- α-DTBZ are completely overlapped with the peak shape of reaction raw materials α-DTBZ,
Inferior separating effect；The retention time of 9-DM- α-DTBZ is 6.60min, and the retention time of α-DTBZ is 6.50min, N- methylbenzenes
The retention time of amine is 9.96min.
Obviously, above-described embodiment is only intended to clearly illustrate example, and not to the restriction of implementation method.It is right
For those of ordinary skill in the art, can also make on the basis of the above description other multi-forms change or
Change.There is no need and unable to be exhaustive to all of implementation method.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (9)
1. the method for separating and assaying of a kind of 9- demethyls-α-dihydrotetrabenazinein and its impurity, it is characterised in that including following step
Suddenly：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 40~90 to add volume ratio：10~60 acetic acid
Ammonium buffer solution-methyl alcohol is dissolved, and the solution that concentration is 0.5~1.5mg/mL is made, as need testing solution；
It is 40~90 with volume ratio with reverse-phase chromatographic column as chromatographic column according to high performance liquid chromatography：10~60 ammonium acetate buffering
Liquid-methyl alcohol is mobile phase, and flow velocity is 0.5~1.5mL/min, and Detection wavelength is 270~290nm, and column temperature is 20~35 DEG C；
The μ L of the need testing solution 5~15 are taken, high performance liquid chromatograph is injected, determined.
2. the method for separating and assaying of 9- demethyls-α-dihydrotetrabenazinein according to claim 1 and its impurity, its feature
It is 50~75 with volume ratio to be：25~50 ammonium acetate buffer-methyl alcohol is mobile phase.
3. the method for separating and assaying of 9- demethyls-α-dihydrotetrabenazinein according to claim 2 and its impurity, its feature
It is,
It is 70 with volume ratio：30 ammonium acetate buffer-methyl alcohol is mobile phase；Or
It is 50 with volume ratio：50 ammonium acetate buffer-methyl alcohol is mobile phase；Or
It is 75 with volume ratio：25 ammonium acetate buffer-methyl alcohol is mobile phase.
4. 9- demethyls-α-dihydrotetrabenazinein according to claim any one of 1-3 and its separation determination side of impurity
Method, it is characterised in that the concentration of the ammonium acetate buffer is 40~60mM, pH value is 4~5；Preferably, the ammonium acetate delays
The concentration of fliud flushing is 50mM, pH value is 4.5.
5. 9- demethyls-α-dihydrotetrabenazinein and its method for separating and assaying of impurity according to claim 1-4, it is special
Levy and be, the filler of the reverse-phase chromatographic column is octadecylsilane chemically bonded silica；Preferably, the reverse-phase chromatographic column is filled out
Fill the octadecylsilane chemically bonded silica that agent is 5 μm of particle diameter.
6. the method for separating and assaying of 9- demethyls-α-dihydrotetrabenazinein according to claim 5 and its impurity, its feature
It is that the reverse-phase chromatographic column is 5 μm, the Sunfire C18 reverse-phase chromatographic columns of 4.6 × 150mm.
7. 9- demethyls-α-dihydrotetrabenazinein according to claim any one of 1-6 and its separation determination side of impurity
Method, it is characterised in that comprise the following steps：
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 70 to add volume ratio：30 ammonium acetate buffering
Liquid-methyl alcohol is dissolved, and the solution that concentration is 1mg/mL is made, as need testing solution；
According to high performance liquid chromatography, the Sunfire C18 reverse-phase chromatographic columns with 5 μm, 4.6 × 150mm are chromatographic column, with volume ratio
It is 70：30 ammonium acetate buffer-methyl alcohol is mobile phase, and flow velocity is 1mL/min, and Detection wavelength is 280nm, and column temperature is 25 DEG C；
The concentration of the ammonium acetate buffer is 50mM, pH value is 4.5；
The μ L of the need testing solution 10 are taken, high performance liquid chromatograph is injected, determined；Or
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 50 to add volume ratio：50 ammonium acetate buffering
Liquid-methyl alcohol is dissolved, and the solution that concentration is 1.0mg/mL is made, as need testing solution；
According to high performance liquid chromatography, the Sunfire C18 reverse-phase chromatographic columns with 5 μm, 4.6 × 150mm are chromatographic column, with volume ratio
It is 50：50 ammonium acetate buffer-methyl alcohol is mobile phase, and flow velocity is 1mL/min, and Detection wavelength is 280nm, and column temperature is 25 DEG C；
The concentration of the ammonium acetate buffer is 50mM, pH value is 4.5；
The μ L of the need testing solution 10 are taken, high performance liquid chromatograph is injected, determined；Or
Weigh 9- demethyls-α-dihydrotetrabenazinein bulk drug to be measured appropriate, it is 75 to add volume ratio：25 ammonium acetate buffering
Liquid-methyl alcohol is dissolved, and the solution that concentration is 1mg/mL is made, as need testing solution；
According to high performance liquid chromatography, the Sunfire C18 reverse-phase chromatographic columns with 5 μm, 4.6 × 150mm are chromatographic column, with volume ratio
It is 75：25 ammonium acetate buffer-methyl alcohol is mobile phase, and flow velocity is 1mL/min, and Detection wavelength is 280nm, and column temperature is 25 DEG C；
The concentration of the ammonium acetate buffer is 50mM, pH value is 4.5；
The μ L of the need testing solution 10 are taken, high performance liquid chromatograph is injected, determined.
8. 9- demethyls-α-dihydrotetrabenazinein according to claim any one of 1-7 and its separation determination side of impurity
Method, it is characterised in that also including following 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein control
The step of product solution, the preparation of methylphenylamine reference substance solution and measure：
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 0.5~1.5mg/mL, as
9- demethyls-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, the solution that concentration is 0.5~1.5mg/mL is made, as α-dihydro butylbenzene
That piperazine reference substance solution；
The reference substance for taking methylphenylamine is appropriate, is made the solution that concentration is 0.5~1.5mg/mL, is compareed as methylphenylamine
Product solution；
9- demethyls-α-dihydrotetrabenazinein the reference substance solution, the α-dihydrotetrabenazinein reference substance solution are taken respectively
5~15 μ L each with the methylphenylamine reference substance solution, injects high performance liquid chromatograph, determines.
9. the method for separating and assaying of 9- demethyls-α-dihydrotetrabenazinein according to claim 8 and its impurity, its feature
It is, also including following 9- demethyls-α-dihydrotetrabenazinein reference substance solution, α-dihydrotetrabenazinein reference substance solution, N-
The step of preparation of methylaniline reference substance solution and measure：
The reference substance for taking 9- demethyls-α-dihydrotetrabenazinein is appropriate, is made the solution that concentration is 1mg/mL, and first is gone as 9-
Base-α-dihydrotetrabenazinein reference substance solution；
The reference substance for taking α-dihydrotetrabenazinein is appropriate, the solution that concentration is 1mg/mL is made, as α-dihydrotetrabenazinein pair
According to product solution；
The reference substance for taking methylphenylamine is appropriate, the solution that concentration is 1mg/mL is made, as methylphenylamine reference substance solution；
9- demethyls-α-dihydrotetrabenazinein the reference substance solution, the α-dihydrotetrabenazinein reference substance solution are taken respectively
10 μ L each with the methylphenylamine reference substance solution, injects high performance liquid chromatograph, determines.
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|---|---|---|---|---|
| CN101985446A (en) * | 2010-06-10 | 2011-03-16 | 江苏省原子医学研究所 | Method for synthesizing (+/-)-9-O-demethyl-alpha-dihydrotetrabenazine |
| CN103776932A (en) * | 2014-01-27 | 2014-05-07 | 暨明医药科技（苏州）有限公司 | Method for analyzing and separating tetrabenazine and intermediate thereof by HPLC (High Performance Liquid Chromatography) method |
| CN105732613A (en) * | 2016-03-29 | 2016-07-06 | 江苏省原子医学研究所 | Synthesis method of 9-demethyl-(+)-alpha-dihydrotetrabenazine |
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2016
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| CN101985446A (en) * | 2010-06-10 | 2011-03-16 | 江苏省原子医学研究所 | Method for synthesizing (+/-)-9-O-demethyl-alpha-dihydrotetrabenazine |
| CN103776932A (en) * | 2014-01-27 | 2014-05-07 | 暨明医药科技（苏州）有限公司 | Method for analyzing and separating tetrabenazine and intermediate thereof by HPLC (High Performance Liquid Chromatography) method |
| CN105732613A (en) * | 2016-03-29 | 2016-07-06 | 江苏省原子医学研究所 | Synthesis method of 9-demethyl-(+)-alpha-dihydrotetrabenazine |
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