CN115389646A - Method for determining amine substances - Google Patents

Abstract

The invention relates to a method for measuring and controlling amine substances. The method adopts reversed-phase chromatographic separation on a high performance liquid chromatograph and adopts an evaporative light scattering detector to detect amine substances. The method can effectively measure the content of amine substances, and has the characteristics of good specificity, high sensitivity, short analysis time and high accuracy of detection results.

Description

Method for determining amine substances

Technical Field

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting amine substances by using an HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector).

Background

Amine substances, such as ethylenediamine, diethylamine, triethylamine and the like and derivatives thereof, are commonly used solvents in drug synthesis. Ethylenediamine is often used as a cosolvent and a pH regulator in medicine, and the content of ethylenediamine needs to be researched and controlled by adopting a proper method. In the prior literature reports, the content of ethylenediamine is determined by gas chromatography and ion chromatography. However, when the ion chromatography is used for measurement, the problems of relatively high sample introduction repeatability, poor peak pattern, poor durability of a chromatographic column and the like exist, the analysis requirements are difficult to meet, and the ion chromatography is expensive and is not widely popularized at present.

CN111721847A1. Discloses a method for detecting the content of ethylenediamine in a medicine by HPLC, which comprises reacting ethylenediamine with a derivative reagent to prepare a derivative reference solution and a test solution; and detecting the derivative reference substance solution and the derivative test substance solution by using high performance liquid chromatography.

Because the existing methods for detecting amines either adopt gas chromatography or HPLC method of pre-column derivatization, which not only has poor durability, but also has complicated derivatization, a method with good durability, higher sensitivity and no need of derivatization is needed to be developed.

At present, no HPLC-ELSD method for determining these amines has been found. In the process of researching the amines, the inventor finds that ELSD has extremely high sensitivity on the detection of the amines, further develops a method for detecting the amines by HPLC-ELSD, and greatly improves the defects of the existing detection method.

Disclosure of Invention

The invention provides a detection method of amine substances, which comprises the step of detecting the amine substances by adopting an HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector) method. The method improves the defects of the existing detection method.

In one embodiment, the detection method of the amine substance comprises performing detection by HPLC-ELSD, wherein a filler of a chromatographic column is octadecyl bonded silica gel or octyl bonded silica gel, a mobile phase a is a trifluoroacetic acid solution, a mobile phase B is acetonitrile-water-formic acid or acetic acid, and gradient elution is performed.

According to the detection method, the amine substance is selected from one or more of methylamine, ethylamine, ethylenediamine, diethylamine, triethylamine and derivatives thereof, the derivatives are preferably isopropylamine or hydroxyethyl ethylenediamine, and the concentration of trifluoroacetic acid, formic acid or acetic acid in the mobile phase A or the mobile phase B is 0.01-2%.

According to the detection method disclosed by the invention, gradient elution is adopted, the water phase ratio in the gradient is not lower than 60% within 0 to 3min, and the specific gradient change is as follows:

the chromatographic conditions of the detection method of the invention are as follows: the sample introduction amount is 1 to 100 mu l, the flow rate is 0.3 to 1.0ml/min, or the column temperature is 15 to 45 ℃, the chromatographic column is Agilent ZORBAX SB-C18,4.6 x 150mm,3.5 mu m, the mobile phase A is 0.2% trifluoroacetic acid solution, or the mobile phase B is acetonitrile-water-formic acid =850:150:1v/v, wherein the temperature of a drift tube of the evaporative light scattering detector is 60 to 105 ℃, or the flow speed of carrier gas is 1.0 to 3.0L/min.

Preferably, in the detection method of the present invention, the sample injection amount is 10 μ L, the flow rate is 1.0ml/min, the column temperature is 35 ℃, the temperature of the drift tube of the evaporative light scattering detector is 95 ℃, and the flow rate of the carrier gas of the evaporative light scattering detector is 3.0L/min.

In the detection method of the invention, the concentration range of the reference substance solution is 1 mu g/ml-1 mg/ml, and the diluent is water, methanol, acetonitrile, ethanol, isopropanol or a mixture of two or more of the water, the methanol, the acetonitrile, the ethanol and the isopropanol.

In the detection method of the present invention, the reference solution: taking the amine reference substance solution to be detected, and preparing the amine reference substance solution into a solution with the concentration of about 1 mu g/ml-1 mg/ml. Test solution: taking each test sample to be tested, and preparing a solution containing the target amine with the concentration of 1 mu g/ml-1 mg/ml.

And (4) calculating a result:

and calculating a regression equation by using the logarithm value of each amine concentration of the reference substance solution and the logarithm value of the corresponding peak area so as to calculate the content of the amine substance in the substance to be detected.

Through a series of experimental researches, the inventor develops the detection method provided by the invention, and the detection method has the advantages that:

(1) The specificity is good, and the separation degree from the adjacent peak is more than 1.5;

(2) The sensitivity is high, and the detection limit is as low as 1 mu g/ml;

(3) The chromatographic peak shape is good, and the number of theoretical plates is more than 5000;

(3) The recovery rate is high in accuracy, the recovery rate measured by the method is 96% -100%, and the RSD is not more than 5%;

(4) The repeatability is good, and the RSD of continuous sample injection of the method is less than 5 percent.

Drawings

FIG. 1 is a chromatogram of blank solution for determination of ethylenediamine in lipoic acid injection.

FIG. 2 chromatogram of ethylene diamine assay-control solution in lipoic acid injection.

FIG. 3 is a solution spectrum of a sample of 20081800 batches for determination of ethylenediamine in lipoic acid injection.

Detailed Description

The following examples are merely exemplary for further illustration and understanding of the nature of the invention, and are not intended to limit the scope of the invention in any way.

Example 1 determination of ethylene diamine in lipoic acid injection

And (3) chromatographic column: agilent ZORBAX SB-C18, 4.6X 150mm,3.5 μm

Mobile phase A:0.2% trifluoroacetic acid solution

And (3) mobile phase B: acetonitrile-water-carboxylic acid (850

Flow rate: 1.0ml/min

The gradient varies as follows:

column temperature: 35 deg.C

Evaporative light scattering detector drift tube temperature: 95 deg.C

Evaporative light scattering detector carrier gas flow rate: 3.0L/min

Sample injection amount: 10 μ l

Preparing a solution:

blank solution: water (I)

Control solution: a proper amount of ethylenediamine was precisely weighed and diluted with water to prepare solutions containing about 0.032mg,0.064mg and 0.096mg of ethylenediamine per 1 ml.

Test solution: precisely measuring 1ml of lipoic acid injection, placing the lipoic acid injection in a 100ml measuring flask, diluting with water to a scale, and shaking up to obtain a test solution.

The measuring method comprises the following steps:

precisely measuring blank solution, reference solution and test solution 10 μ l each, injecting into high performance liquid chromatograph, and recording chromatogram, the result is shown in figures 1-3. The results are shown in Table 1.

TABLE 1 detection results of ethylenediamine content in three liponic acid injections

EXAMPLE 2 determination of isopropylamine in metoprolol succinate

A chromatographic column: agilent ZORBAX SB-C18, 4.6X 150mm,3.5 μm

A mobile phase A:0.2% trifluoroacetic acid solution

Mobile phase B: acetonitrile-water-carboxylic acid (850

Flow rate: 1.0ml/min

The gradient varies as follows:

column temperature: 30 deg.C

Evaporative light scattering detector drift tube temperature: 80 deg.C

Evaporative light scattering detector carrier gas flow rate: 2.0L/min

Sample introduction amount: 20 μ l

Preparing a solution:

control solution: an appropriate amount of isopropylamine is weighed precisely and diluted with water to prepare solutions containing isopropylamine 0.005mg, isopropylamine 0.010mg and isopropylamine 0.020mg in each 1 ml.

100% of the standard sample solution: accurately weighing a proper amount of metoprolol succinate and isopropylamine, dissolving with water and diluting to prepare a solution containing 100mg of metoprolol succinate and 0.010mg of isopropylamine per 1 ml.

The determination method comprises the following steps:

precisely measuring the reference solution and 100% standard sample solution by 20 μ l each, injecting into high performance liquid chromatograph, and recording chromatogram. The results are shown in Table 2.

TABLE 2 detection results of the content of isopropylamine in metoprolol succinate

Example 3 determination of hydroxyethylethylenediamine in mitoxantrone hydrochloride

A chromatographic column: agilent XDB-C8, 4.6X 150mm,5 μm

A mobile phase A:0.1% trifluoroacetic acid solution

Mobile phase B: methanol-water-acetic acid (700

Flow rate: 1.0ml/min

The gradient varies as follows:

column temperature: 30 deg.C

Evaporative light scattering detector drift tube temperature: 105 ℃ C

Evaporative light scattering detector carrier gas flow rate: 3.0L/min

Sample introduction amount: 5 μ l

Preparing a solution:

control solution: an appropriate amount of hydroxyethylethylenediamine was weighed out precisely and diluted with water to give solutions containing hydroxyethylethylenediamine in an amount of about 0.009mg,0.018mg and 0.036mg per 1 ml.

100% of the standard sample solution: a proper amount of mitoxantrone hydrochloride and hydroxyethyl ethylenediamine are precisely weighed, dissolved and diluted with water to prepare a solution containing about 100mg of mitoxantrone hydrochloride and about 0.018mg of hydroxyethyl ethylenediamine per 1 ml.

The determination method comprises the following steps:

precisely measuring the reference solution and 100% standard sample solution by 5 μ l each, injecting into high performance liquid chromatograph, and recording chromatogram. The results are shown in Table 3.

TABLE 3 detection results of hydroxyethylethylenediamine content in mitoxantrone hydrochloride

Example 4 method validation

A chromatographic column: agilent ZORBAX SB-C18, 4.6X 150mm,3.5 μm

A mobile phase A:0.2% trifluoroacetic acid solution

Mobile phase B: acetonitrile-water-carboxylic acid (850

Flow rate: 1.0ml/min

The gradient varies as follows:

column temperature: 35 deg.C

Evaporative light scattering detector drift tube temperature: 95 deg.C

Evaporative light scattering detector carrier gas flow rate: 3.0L/min

Sample introduction amount: 10 μ l

1. Specificity and System suitability test

Precisely measuring blank solution (water), reference solution and sample solution 10 μ l each, injecting into high performance liquid chromatograph, and recording chromatogram, the results are shown in Table 4.

TABLE 4 specificity and System suitability test results

The results show that: the blank solution does not interfere the component measurement, and the ethylene diamine in the reference solution and the test solution has good peak type and does not interfere the measurement by other impurity peaks; the control solution was injected continuously for 6 needles, and the peak area logarithm RSD was 0.2%.

2. Linear test

Mu.l of each 1ml of the solutions containing about 32. Mu.g, 51.2. Mu.g, 64. Mu.g, 96. Mu.g and 128. Mu.g of ethylenediamine was precisely measured, and the solutions were injected into a high performance liquid chromatograph and the chromatogram was recorded. The regression equation was calculated from the logarithmic value of the ethylenediamine concentration in the control solution and the logarithmic value of the corresponding peak area, and the results are shown in table 5.

TABLE 5 logarithmic value of concentration and logarithmic value of peak area of ethylenediamine in control solution

The results show that: the linear equation of the ethylenediamine is lg (A) =1.3392 Xlg (C) +3.6993, the correlation coefficient is 0.999, and the linear relation is good within the range of 31.81 mu g/ml to 127.22 mu g/ml.

3. Accuracy test

Precisely measuring 10 μ l of test solution containing ethylenediamine 80%, 100% and 120%, respectively, injecting into high performance liquid chromatograph, and recording chromatogram. The results are shown in Table 6.

TABLE 6 detection results of ethylenediamine test solution

The results show that: the recovery rate of the ethylenediamine is within the range of 96-100%, and the RSD is 2%. The method has high accuracy, and can accurately detect the content of the ethylenediamine in the test sample.

4. Precision test

Different personnel respectively measure 10 mul of the sample solution with precision at different time, inject the sample solution into a high performance liquid chromatograph, and record chromatogram. The results are shown in Table 7.

TABLE 7 test results of two analysts who completed the experimental operation individually

The results show that: different personnel test that the content of the ethylenediamine in 6 parts of the test solution is within the range of 96-102%, the RSD is 2%, and the RSD of the content of the ethylenediamine in 12 parts of the test solution is 3%. The method has good precision.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can be covered within the technical scope of the present invention without any changes and substitutions that may occur in the inventive idea.

Claims (12)

1. A detection method of amine substances comprises the step of detecting by adopting HPLC-ELSD, wherein a filler of a chromatographic column is octadecyl bonded silica gel or octyl bonded silica gel, a mobile phase A is trifluoroacetic acid solution, a mobile phase B is acetonitrile-water-formic acid or acetic acid, and gradient elution is adopted.

2. The process according to claim 1, the amine species being selected from one or more of methylamine, ethylamine, ethylenediamine, diethylamine, triethylamine and derivatives thereof, preferably isopropylamine or hydroxyethylethylenediamine.

3. The detection method according to claim 1, wherein the concentration of trifluoroacetic acid, formic acid or acetic acid in the mobile phase A or the mobile phase B is 0.01% -2%.

4. The detection method according to claim 1, wherein the gradient elution is performed, and the water ratio in the gradient is not lower than 60% within 0 to 3 min.

5. The detection method according to claim 4, wherein the gradient is eluted and the gradient is changed as follows:

。

6. the detection method according to claim 1, wherein the chromatographic conditions are as follows: the sample introduction amount is 1 to 100 mu l, the flow rate is 0.3 to 1.0ml/min, or the column temperature is 15 to 45 ℃.

7. The detection method according to claim 1, wherein the ELSD and the evaporative light scattering detector drift tube have a temperature of 60 to 105 ℃ or a carrier gas flow rate of 1.0 to 3.0L/min.

8. The detection method according to claim 1, wherein the concentration of the control solution is in the range of 1 μ g/ml to 1mg/ml.

9. The detection method according to claim 1 to 8, wherein the diluent is water, methanol, acetonitrile, ethanol, isopropanol or a mixture of two or more thereof.

10. The detection method according to claim 1, wherein the chromatographic column is Agilent ZORBAX SB-C18,4.6 x 150mm,3.5 μm.

11. The assay of claim 1, wherein mobile phase a is a 0.2% trifluoroacetic acid solution and mobile phase B is acetonitrile-water-formic acid =850:150:1v/v.

12. The detection method according to claim 6 or 7, wherein the sample introduction amount is 10 μ L, the flow rate is 1.0ml/min, the column temperature is 35 ℃, the evaporative light scattering detector drift tube temperature is 95 ℃, and the evaporative light scattering detector carrier gas flow rate is 3.0L/min.

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