A method for determining impurities in dalbavancin

Abstract

The invention discloses a method for detecting impurities in dalbavancin hydrochloride. The invention adopts a pre-column derivatization reverse high performance liquid chromatography method to detect 3-dimethylaminopropylamine in dalbavancin hydrochloride. The detection method of the invention is suitable for detecting the content of 3-dimethylaminopropylamine in the acidic compound dalbavancin, and the method has strong versatility, good system durability, good stability and low detection cost.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to an analytical method for determining 3-dimethylaminopropylamine in dalbavancin hydrochloride. Background Art

[0002] Dalbavancin hydrochloride is the hydrochloride of a mixture of dalbavancin A0, A1, B0, B1 and B2, wherein dalbavancin B0 is the main component. These five components have the same parent core structure, and the structural formula is as follows. The chemical formula and molecular weight of each component are shown in Table 1:

[0003]

[0004] Dalbavancin <![CDATA[R1]]> <![CDATA[R2]]> Molecular formula Molecular weight <![CDATA[A0]]> <![CDATA[CH(CH3)2]]> H <![CDATA[C 87 H 98 N 10 O 28 Cl2]]> 1802.7 <![CDATA[A1]]> <![CDATA[CH2CH2CH3]]> H <![CDATA[C 87 H 98 N 10 O 28 Cl2]]> 1802.7 <![CDATA[B0]]> <![CDATA[CH2CH(CH3)2]]> H <![CDATA[C 88 H 100 N 10 O 28 Cl2]]> 1816.7 <![CDATA[B1]]> <![CDATA[CH2CH2CH2CH3]]> H <![CDATA[C 88 H 100 N 10 O 28 Cl2]]> 1816.7 <![CDATA[B2]]> <![CDATA[CH2CH(CH3)2]]> <![CDATA[CH3]]> <![CDATA[C 89 H 102 N 10 O 28 Cl2]]> 1830.7

[0005] 3-Dimethylaminopropylamine is used as a starting material in the synthesis of dalbavancin and may remain in the final product to a certain extent.

[0006] 3-Dimethylamino-1-propylamine, also known as N,N-dimethyl-1,3-propylenediamine (English name N,N'-Dimethyl-1,3-propyldiamine), or 3-dimethylamino-n-propylamine, is a low-order aliphatic diamine. It contains both tertiary and primary amine groups in the molecule. It is a colorless, transparent liquid with an ammonia smell. It emits smoke in the air and is soluble in water and organic solvents. The molecular formula is C5H 14 N2, with the structural formula (CH3)2NCH2CH2CH2NH2. Its main use is as a raw material for the synthesis of pesticides and surfactants. 3-Dimethylaminopropylamine is a highly toxic compound that has a strong irritating effect on the skin and can cause severe poisoning if inhaled or taken orally. However, its limit is not clearly defined. According to the definition of unknown impurity limits in ICH, its limit is controlled below 0.1%.

[0007] The Chinese patent "A method for determining impurities in dalbavancin" (application number 201710238376.X) discloses a detection method for determining 3-dimethylaminopropylamine residues in dalbavancin hydrochloride. The method uses a strong alkaline solution to dissolve the acidic compound dalbavancin, and replaces and releases the 3-dimethylaminopropylamine bound to the salt to form 3-dimethylaminopropylamine free base. The phenomenon of stratification caused by the immiscibility of the strong polar solvent acetonitrile and the alkaline solution is utilized, and acetonitrile is used to perform liquid-liquid extraction of the highly polar 3-dimethylaminopropylamine in the strong alkaline solution of dalbavancin, and saturated inorganic salts are added to enhance the extraction effect of acetonitrile. A flame ionization detector is used, and the acetonitrile layer (upper layer) is sampled by direct injection gas chromatography, thereby avoiding strong alkali from entering the chromatographic system to damage the system. The present invention finds a reversed-phase high performance liquid chromatography method with a different principle, which has strong versatility, good system durability, good stability, is suitable for the detection method of 3-dimethylaminopropylamine content in the acidic compound dalbavancin, and has low detection cost.

[0008] Due to the high requirements of drugs on the purity of the final product, the residual content of 3-dimethylaminopropylamine is very low. When the conventional analytical method acid-base titration is used for determination, the sensitivity and accuracy of the general titration method cannot meet the requirements.

[0009] Since 3-dimethylaminopropylamine has a weak ultraviolet response, a pre-column derivatization high performance liquid chromatography method is used to control 3-dimethylaminopropylamine. 3-dimethylaminopropylamine belongs to amines, and a derivative is obtained by an amide derivatization reaction with a derivatizing agent. Then, a chromatographic column with an octadecylsilane bonded phase as the stationary phase is used, and the mobile phase is composed of phase A and phase B, wherein a mixture of a buffered salt solution and an organic solvent is used as phase A, and an organic solvent is used as phase B; gradient elution is performed in a liquid chromatography system, and a spectrum is recorded. Summary of the invention

[0010] The invention is a new method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride, and provides a new option for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride.

[0011] The present invention provides a method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride, comprising the following steps:

[0012] A. Preparation of test solution: Take dalbavancin hydrochloride test sample, weigh accurately, dissolve in 50-70% acetonitrile aqueous solution and quantitatively dilute to a solution containing 4-6 mg per 1 ml, shake well and set aside.

[0013] B. Preparation of reference solution: Take 3-dimethylaminopropylamine reference substance, weigh accurately, dissolve in acetonitrile and quantitatively dilute to a solution containing 0.04mg~0.06mg per 1ml; accurately transfer 1ml of the above solution to a 10ml volumetric flask, dilute to the mark with 50~70% acetonitrile aqueous solution, shake well and set aside.

[0014] C. Preparation of derivatization agent: Take benzoyl chloride, weigh accurately, add acetonitrile to dissolve and quantitatively dilute to a solution containing 2mg~3mg per 1ml, shake well and set aside.

[0015] D. Derivatization process: Accurately pipette 1 ml of each of 75% acetonitrile aqueous solution, reference solution and test solution into a 5 ml volumetric flask, pipette 1 ml of borate buffer and derivatization agent in sequence, shake for 1 min, pipette 1 ml of ammonia water, shake for 1 min, dilute to the mark with purified water, and shake well. Heat in a 60°C water bath for 60 min, take out and cool for later use.

[0016] E. Detect by high performance liquid chromatography and calculate the content of 3-dimethylaminopropylamine by peak area according to the external standard method.

[0017] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the steps A and B, the dissolving liquid in the last dilution step is a 50-70% acetonitrile aqueous solution, preferably a 60% acetonitrile aqueous solution.

[0018] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the steps A, B, and C, the concentrations of the test sample, the reference sample, and the derivatizing agent are configured based on the molar ratio of the total amount of 3-dimethylaminopropylamine and dalbavancin hydrochloride to the amount of the derivatizing agent used being 1:4 to 6, preferably 1:5.

[0019] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the step C, the derivatizing agent is benzoyl chloride.

[0020] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the step D, borate buffer: 0.05 M / L borax aqueous solution-0.2 M / L boric acid aqueous solution=5.5:4.5 (v / v).

[0021] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the step D, the purpose of adding ammonia water is to neutralize the excess solvent of the derivatizing agent.

[0022] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the step D, the purpose of water bath heating is to promote the decomposition of excess derivatizing agent, the temperature is 60-65° C., the time is 60-80 min, preferably 60° C., 60 min.

[0023] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the step E, the liquid chromatograph model is Agilent 1260 or Shimadzu LC2030 C, and the chromatographic column can be a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, with a column length of 250 mm, a column inner diameter of 4.6 mm, and a particle size of 5 µm, preferably Ultimate ® C18, 4.6 mm×250 mm, 5 µm chromatographic column.

[0024] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the step E, the liquid chromatography conditions are:

[0025] Detection wavelength: 230nm

[0026] Flow rate: 1.0ml / min

[0027] Column temperature: 40°C

[0028] Injection volume: 50 μl

[0029] Mobile phase A: 0.02 mol / L sodium dihydrogen phosphate. Dihydrate pH = 2.1 (pH adjusted with phosphoric acid)

[0030] Mobile phase B: acetonitrile

[0031] Furthermore, the method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride is characterized in that in the step E, the elution procedure of mobile phases A and B is:

[0032] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 85 15 15 20 80 20 20 80 21 90 10 25 90 10

[0033] 3-Dimethylaminopropylamine has a weak ultraviolet response, so a pre-column derivatization high performance liquid chromatography method is used to control 3-dimethylaminopropylamine. 3-Dimethylaminopropylamine belongs to amines, and a derivative is obtained by an amide derivatization reaction between a derivatizing agent and the amine. Then, a chromatographic column with an octadecylsilane bonded phase as the stationary phase is used, and the mobile phase is composed of phase A and phase B, wherein a mixture of a buffered saline solution and an organic solvent is used as phase A, and an organic solvent is used as phase B; gradient elution is performed in a liquid chromatography system, and a spectrum is recorded. The present invention provides a new analytical method for determining 3-dimethylaminopropylamine, an impurity of dalbavancin. The analytical method of the present invention has high sensitivity, few interfering peaks, good peak shape, low detection cost, high sensitivity, strong versatility, and significant progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The HPLC chart of Example 1 is the derivatization spectrum of the test sample. Under the derivatization conditions, there is an interference peak at the elution peak of the 3-dimethylaminopropylamine derivative in the chart.

[0035] Figure 2 The HPLC chart of Example 2 is the derivatization spectrum of the test sample. Under the derivatization conditions, there is no interfering peak at the elution point of the 3-dimethylaminopropylamine derivative in the chart.

[0036] Figure 3 The HPLC chart of Example 3 is the spectrum of the reference solution of derivatization process 1. The peak in the chart is the peak of the 3-dimethylaminopropylamine derivative. Under the derivatization conditions, an interfering peak exists at the elution point of the 3-dimethylaminopropylamine derivative.

[0037] Figure 4 The HPLC chart of Example 3 is the spectrum of the reference solution of derivatization process 2. The peak in the chart is the peak of the 3-dimethylaminopropylamine derivative. Under the derivatization conditions, there is no interfering peak at the elution point of the 3-dimethylaminopropylamine derivative.

[0038] Figure 5 , the HPLC chart of Example 4, the peak in the chart is the peak of the 3-dimethylaminopropylamine derivative. Under this chromatographic condition, the peak shape of the 3-dimethylaminopropylamine derivative is relatively good.

[0039] Figure 6 , the HPLC chart of Example 5, the peak in the chart is the peak of the 3-dimethylaminopropylamine derivative. Under the chromatographic conditions, the peak shape of the 3-dimethylaminopropylamine derivative is poor.

[0040] Figure 7 , the HPLC chart of Example 6, the peak in the chart is the peak of the 3-dimethylaminopropylamine derivative. Under the chromatographic conditions, the peak shape of the 3-dimethylaminopropylamine derivative is poor.

[0041] Figure 8 , the HPLC chart of Example 7, the peak in the chart is the peak of the 3-dimethylaminopropylamine derivative, and under the chromatographic conditions, there is an interfering peak at the elution point of the 3-dimethylaminopropylamine derivative.

[0042] Fig. 9 , the HPLC chart of Example 8, the peak in the chart is the peak of the 3-dimethylaminopropylamine derivative. Under the chromatographic conditions, the peak shape of the 3-dimethylaminopropylamine derivative is very good. Example

[0043] Example 1

[0044] High performance liquid chromatograph: LC-2030C

[0045] Chromatographic column: Octadecylsilane bonded silica column (Ultimate ® C18, 4.6mm×250mm, 5 µm)

[0046] Mobile phase: 0.02 mol / L sodium dihydrogen phosphate dihydrate aqueous solution as mobile phase A; acetonitrile as mobile phase B

[0047] The elution procedures of mobile phases A and B are:

[0048] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 85 15 15 20 80 20 20 80 21 90 10 25 90 10

[0049] Flow rate: 1.0ml / min

[0050] Wavelength: 210nm

[0051] Injection volume: 50ul

[0052] Column temperature: 40°C

[0053] Implementation steps:

[0054] Boric acid-borax buffer: 0.05 mol / L borax aqueous solution-0.2 mol / L boric acid aqueous solution = 55:45 (v / v) Derivatization reagent: 5 mg / ml fluorenylmethoxycarbonyl chloride acetonitrile solution

[0055] Control solution: accurately weigh about 50 mg of 3-dimethylaminopropylamine into a 10 ml volumetric flask, dissolve it in acetonitrile and dilute to the mark, shake well, and use it as the 3-dimethylaminopropylamine control stock solution; accurately transfer 1 ml of the 3-dimethylaminopropylamine control stock solution into a 100 ml volumetric flask, dilute it to the mark with acetonitrile, shake well, and use it as the 3-dimethylaminopropylamine control primary stock solution; accurately transfer 1 ml of the 3-dimethylaminopropylamine control primary stock solution into a 10 ml volumetric flask, dilute it to the mark with 75% acetonitrile, shake well, and use it as the 3-dimethylaminopropylamine control primary stock solution.

[0056] Test solution: Accurately weigh about 50 mg of the test sample into a 10 ml volumetric flask, dissolve it in 75% acetonitrile aqueous solution and dilute to the scale and shake well.

[0057] Derivatization process: Accurately measure 1 ml of 75% acetonitrile aqueous solution, control solution and test solution into 5 ml volumetric flasks respectively, add 1 ml of boric acid-borax buffer, then add 1 ml of derivatization reagent, shake for 1 min, add 1 ml of ammonia water, shake for another 1 min, dilute to the scale with purified water, and shake well.

[0058] Take 50 μl of blank, reference and test sample derivatization solutions and inject them into the liquid chromatograph to record the chromatogram. Figure 1 .

[0059] Example 2

[0060] High performance liquid chromatograph: Agilent 1260

[0061] Derivatization reagent: 2.5 mg / ml benzoyl chloride acetonitrile solution;

[0062] The remaining conditions are the same as in Example 1;

[0063] Implementation steps:

[0064] Take 50 μl of blank, reference and test sample derivatization solutions and inject them into the liquid chromatograph to record the chromatogram. Figure 2 .

[0065] Example 3

[0066] High performance liquid chromatograph: Agilent 1260

[0067] Chromatographic column: octadecylsilane bonded silica gel column (Ultimate ® C18, 4.6mm×250mm, 5 µm) Mobile phase: 0.02mol / L sodium dihydrogen phosphate dihydrate aqueous solution as mobile phase A; acetonitrile as mobile phase B. The elution procedures of mobile phases A and B are:

[0068] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 85 15 15 20 80 20 20 80 21 90 10 25 90 10

[0069] Flow rate: 1.0ml / min

[0070] Wavelength: 265nm

[0071] Injection volume: 50ul

[0072] Column temperature: 40°C

[0073] Implementation steps:

[0074] Boric acid-borax buffer solution: 0.05 mol / L borax aqueous solution-0.2 mol / L boric acid aqueous solution = 55:45 (v / v)

[0075] Derivatization reagent: 2.5 mg / ml benzoyl chloride acetonitrile solution

[0076] Control solution: accurately weigh about 50 mg of 3-dimethylaminopropylamine into a 10 ml volumetric flask, dissolve it in acetonitrile and dilute to the mark, shake well, and use it as the 3-dimethylaminopropylamine control stock solution; accurately transfer 1 ml of the 3-dimethylaminopropylamine control stock solution into a 100 ml volumetric flask, dilute it to the mark with acetonitrile, shake well, and use it as the 3-dimethylaminopropylamine control primary stock solution; accurately transfer 1 ml of the 3-dimethylaminopropylamine control primary stock solution into a 10 ml volumetric flask, dilute it to the mark with 60% acetonitrile, shake well, and use it as the 3-dimethylaminopropylamine control primary stock solution.

[0077] Derivatization process 1: Accurately measure 1 ml of 60% acetonitrile aqueous solution and control solution in 5 ml volumetric bottles, add 1 ml of boric acid-borax buffer, add 1 ml of derivatization reagent, shake for 1 min, add 1 ml of ammonia water, shake for 1 min, dilute to the mark with purified water, shake well. Heat in a 40°C water bath for 60 min, take out and cool.

[0078] Derivatization process 2: Accurately measure 1 ml of 60% acetonitrile aqueous solution and control solution in 5 ml volumetric bottles, add 1 ml of boric acid-borax buffer, add 1 ml of derivatization reagent, shake for 1 min, add 1 ml of ammonia water, shake for 1 min, dilute to the mark with purified water, shake well. Heat in a 60°C water bath for 60 min, take out and cool.

[0079] Take 50 μl of the blank derivatization solution and the reference derivatization solution from derivatization process 1 and 2 respectively and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the attached Figure 3 , 4 .

[0080] Example 4

[0081] Mobile phase: 0.02 mol / L sodium dihydrogen phosphate dihydrate aqueous solution is mobile phase A;

[0082] The remaining conditions are the same as in Example 1;

[0083] Implementation steps:

[0084] Take 50 μl of the blank derivatization solution and the reference derivatization solution of Derivatization Process 2 in Example 3 and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the attached Figure 5 .

[0085] Example 5

[0086] Mobile phase: 0.01 mol / L sodium dihydrogen phosphate dihydrate aqueous solution is mobile phase A;

[0087] Wavelength: 230nm

[0088] The remaining conditions are the same as those in Example 3;

[0089] Implementation steps:

[0090] Take 50 μl of the blank derivatization solution and the reference derivatization solution of Derivatization Process 2 in Example 3 and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the attached Figure 6 .

[0091] Example 6

[0092] Mobile phase: 0.02 mol / L sodium dihydrogen phosphate dihydrate aqueous solution (pH adjusted to 5.5 with sodium hydroxide) as mobile phase A;

[0093] The remaining conditions are the same as in Example 5;

[0094] Implementation steps:

[0095] Take 50 μl of the blank derivatization solution and the reference derivatization solution of Derivatization Process 2 in Example 3 and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the attached Figure 7 .

[0096] Example 7

[0097] Mobile phase: 0.02 mol / L sodium dihydrogen phosphate dihydrate aqueous solution (pH adjusted to 3.5 with phosphoric acid) as mobile phase A;

[0098] The remaining conditions are the same as in Example 5;

[0099] Implementation steps:

[0100] Take 50 μl of the blank derivatization solution and the reference derivatization solution of Derivatization Process 2 in Example 1 and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the attached Figure 8 .

[0101] Example 8

[0102] Mobile phase: 0.02 mol / L sodium dihydrogen phosphate dihydrate aqueous solution (pH adjusted to 2.1 with phosphoric acid) as mobile phase A;

[0103] The remaining conditions are the same as in Example 5;

[0104] Implementation steps:

[0105] Take 50 μl of the blank derivatization solution and the reference derivatization solution of Derivatization Process 2 in Example 3 and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the attached Fig. 9 .

[0106] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for detecting 3-dimethylaminopropylamine in dalbavancin hydrochloride, comprising the following steps: A. Preparation of test solution: Take dalbavancin hydrochloride test sample and prepare a dilution for later use; B, reference solution preparation: Take 3-dimethylaminopropylamine reference substance, prepare a dilution solution for standby use; C, derivatization agent solution preparation: accurately weigh the derivatization agent benzoyl chloride and prepare a dilution; D. Derivatization process: Accurately pipette 1 ml of 50-70% acetonitrile aqueous solution, reference solution and test solution into a 5 ml volumetric flask, pipette 1 ml of borate buffer and derivatization agent solution into the flask in sequence, shake for 1 min, continue to pipette 1 ml of ammonia water into the flask, shake for 1 min, dilute to the mark with purified water, and shake well; heat in a water bath at 55-65°C for 50-70 min, take out and cool for later use; E. Detect by high performance liquid chromatography, and calculate the content of 3-dimethylaminopropylamine by peak area according to the external standard method; In the step E, the liquid chromatograph model is Agilent 1260 or Shimadzu LC2030C, and the chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, with a column length of 250 mm, a column inner diameter of 4.6 mm, and a particle size of 5 µm; In the step E, the liquid chromatography conditions are: Detection wavelength: 230nm Flow rate: 1.0ml / min Column temperature: 40°C Injection volume: 50 μl Mobile phase A: 0.02 mol / L sodium dihydrogen phosphate dihydrate, pH = 2.1 Mobile phase B: acetonitrile; In the step E, the elution procedure of mobile phases A and B is: 。 2. The method according to claim 1, characterized in that The preparation method of the diluent in step A is as follows: take the dalbavancin hydrochloride test sample, accurately weigh it, add a 50-70% by volume acetonitrile aqueous solution to dissolve it and quantitatively dilute it into a solution containing 4 mg to 6 mg per 1 ml, shake it well and set it aside.

3. The method according to claim 1, characterized in that The preparation method of the diluent in step B is as follows: take a 3-dimethylaminopropylamine reference substance, accurately weigh it, add acetonitrile to dissolve it and quantitatively dilute it into a solution containing 0.04mg~0.06mg per 1ml; accurately transfer 1ml of the diluted solution to a 10ml volumetric bottle, add a 50~70% acetonitrile aqueous solution by volume to dilute it to the scale, and shake it for later use.

4. The method according to any one of claims 1 to 3, characterized in that The preparation method of the derivatizing agent solution in step C is as follows: accurately weigh benzoyl chloride, add acetonitrile to dissolve and quantitatively dilute to a solution containing 2 mg to 3 mg per 1 ml, and shake well for later use.

5. The method according to claim 1, characterized in that In the steps A, B and C, the concentrations of the test sample, reference sample and derivatizing agent are configured based on the molar ratio of the total amount of 3-dimethylaminopropylamine and dalbavancin hydrochloride to the amount of the derivatizing agent being 1:4-6.

6. The method according to claim 1, characterized in that In the step D, the borate buffer: 0.05M / L borax aqueous solution - 0.2M / L boric acid aqueous solution = 5.5:4.5 (v / v).

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