CN117405796A

CN117405796A - Method for simultaneously detecting multiple short-chain fatty amines and application

Info

Publication number: CN117405796A
Application number: CN202311460774.8A
Authority: CN
Inventors: 朱婧; 罗琼枝; 刘秀; 王玲兰; 龚翠云; 朱维君
Original assignee: Hunan Qianjin Xiangjiang Pharmaceutical Co ltd; Qianjin Pharmaceutical Co ltd
Current assignee: Hunan Qianjin Xiangjiang Pharmaceutical Co ltd; Qianjin Pharmaceutical Co ltd
Priority date: 2023-11-03
Filing date: 2023-11-03
Publication date: 2024-01-16

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously detecting multiple short-chain fatty amines and application thereof. According to the method, after derivatization treatment is carried out on a sample to be detected by using the halogenated nitrobenzene compound, a substance with color is formed, so that false negative caused by esterification reaction of the chain fatty amine can be avoided, the response value of the chain fatty amine can be increased, the peak value is improved, the active ingredients and various short chain fatty amines in the bulk drug and the like can be detected simultaneously under the same chromatographic condition, the separation effect is good, the sensitivity of the method is high, the solution stability is good, the specificity meets the requirements, the product quality of the bulk drug or the finished drug can be better controlled, and the safety is improved.

Description

Method for simultaneously detecting multiple short-chain fatty amines and application

Technical Field

The invention belongs to the technical field of analysis and detection. More particularly, to a method for simultaneously detecting a plurality of short chain fatty amines and application thereof.

Background

Short-chain fatty amines such as ammonia, methylamine and dimethylamine are common materials in the synthesis of raw materials, are harmful impurities, and have great potential safety hazards when residues remain in the medicines. For example, in the synthesis route of Fu Nuola fumaric acid, methylamine is required to be used for methylamine, ammonia and dimethylamine are contained in the methylamine, and the ammonia and the dimethylamine can synchronously participate in the reaction to generate corresponding organic impurities, so that in order to ensure the quality control of the voronoi fumaric acid, an analysis method for simultaneously detecting the ammonia, the methylamine and the dimethylamine is required to be established, the residue of the ammonia, the methylamine and the dimethylamine is controlled, and the removal condition of related impurities is studied. Other crude drugs requiring synthesis of short chain fatty amines such as ammonia, methylamine, dimethylamine and the like have similar conditions, so that there is an urgent need to establish a simple method for simultaneously measuring the residual amounts of 3 fatty amines in the crude drugs or intermediate products thereof.

Because the short-chain aliphatic amines such as ammonia, methylamine, dimethylamine and the like have simple and similar structures, have no strong-absorption ultraviolet functional groups and have no response by adopting a common high-efficiency liquid phase method. At present, a gas chromatography method is often adopted to carry out single detection on short-chain fatty amine such as ammonia, methylamine or dimethylamine, for example, chinese patent application CN108226338A discloses a method for detecting the dimethylamine containing the intermediate of the sarpogrelate hydrochloride, the method adopts the gas chromatography to carry out detection, and when only the dimethylamine exists, the method has good precision, accuracy, reproducibility and durability, but when the ammonia and the methylamine exist, the gas chromatography can not well separate the ammonia, the methylamine and the dimethylamine, and the problems of poor peak type, low response and the like exist; and most of the crude drugs possibly contain acidic substances such as fumaric acid, maleic acid, hydrochloric acid and the like, for example, by adopting a direct sample preparation method, the acidic substances can react with amines to generate esters, and the difficulty of detecting amine impurities in the crude drugs and intermediate products is further increased.

Disclosure of Invention

The invention aims to solve the technical problems that the existing gas chromatography can not separate various short-chain fatty amines well and has poor peak type, low response and the like; the method is simple to operate, high in sensitivity and good in stability.

The invention aims to provide application of the method for simultaneously detecting a plurality of short-chain fatty amines in medicine quality monitoring.

The above object of the present invention is achieved by the following technical scheme:

a method for simultaneously detecting a plurality of short-chain aliphatic amines comprises the steps of firstly treating a sample to be detected with a catalyst organic amine compound and a derivatization agent halogenated nitrobenzene compound, and then detecting according to the following high performance liquid chromatography conditions:

chromatographic column: octadecylsilane chemically bonded silica packed column, detection wavelength: 400-430 nm, column temperature: the temperature is 35-50 ℃ and the flow rate is 0.9-1.1 ml/min; mobile phase a:0.05 to 0.15 percent formic acid aqueous solution, mobile phase B: methanol and acetonitrile (25-40): the mixed solution of (60-75) was tested under the following gradient elution conditions:

according to the invention, after the sample to be detected is subjected to derivatization treatment by using the catalyst organic amine compound and the derivatization agent halogenated nitrobenzene compound, a substance with color is formed, so that false negative caused by the esterification reaction of the chain fatty amine can be avoided, the response value of the chain fatty amine can be increased, the peak value is improved, and the absorption wavelength is effectively transferred to a visible region (400-450 nm). On the other hand, most of the raw materials, impurities and the derivative reagent adopted by the invention are very weak in absorption, and the quantitative limit and the detection limit can be obviously reduced due to the red shift effect and the interference minimization.

Wherein R1 and R2 are each independently selected from-H, -CH ₃ 、C ₂ H ₆ 、C ₃ H ₉ And short chain fatty alkanes.

Preferably, the detection wavelength is preferably 416nm.

Further, the treatment temperature of the catalyst organic amine compound and the derivatization agent halogenated nitrobenzene compound is 55-85 ℃. Preferably, the treatment temperature of the catalyst organic amine compound and the derivatization agent halogenated nitrobenzene compound is 70-80 ℃.

Further, the treatment time of the catalyst organic amine compound and the derivatization agent halogenated nitrobenzene compound is 45-120 min. Preferably, the treatment time of the catalyst organic amine compound and the derivatization agent halogenated nitrobenzene compound is 90-120 min.

Further, the derivatizing agent halogenated nitrobenzene compound comprises 4-fluoro-3-nitrobenzotrifluoride, 1-fluoro-2-nitrobenzene, 1-fluoro-4-nitrobenzene or 2, 4-dinitrobenzene.

Further, the catalyst organic amine compound comprises tertiary amine such as N, N-diisopropylethylamine, triethylamine and the like. Care should be taken in the selection to avoid introducing fatty amines which may participate in the reaction, so as not to cause interference or false positive results.

Preferably, the addition amounts of the catalyst organic amine compound and the derivatizing agent halogenated nitrobenzene compound are determined according to the content of the short-chain fatty amine in the sample to be detected, and the short-chain fatty amine in the sample to be detected needs to be completely derivatized. For example, when the sample to be detected is a vonolamine fumarate bulk drug, the impurity content is low, and the addition amount of the derivative agent halogenated nitrobenzene compound is 400-500 mu L; when the sample to be detected is methylamine, the impurity content is more, and the addition amount of the derivatization agent halogenated nitrobenzene compounds is 4.8-5.2 ml.

Further, the column was Phenomenex Ultremex u C18, 4.6mm.times.250 mm,5 μm.

Further, the sample injection amount was 5. Mu.l when the HPLC was performed.

Further, the short-chain fatty amine is fatty amine with carbon number less than 4, including ammonia, methylamine, dimethylamine, ethylamine, diethylamine, propylamine or n-butylamine. The method has wide applicability and strong specificity.

Further, the limit of detection of ammonia was 0.002%, the limit of detection of methylamine was 0.003%, and the limit of detection of dimethylamine was 0.01%.

Further, the quantitative limit of ammonia or methylamine is 0.01%, and the quantitative limit of dimethylamine is 0.03%.

The method has high sensitivity.

In addition, the invention also claims the application of the method for simultaneously detecting various short-chain fatty amines in the quality monitoring of bulk drugs or finished drugs.

Further, at least one impurity selected from ammonia, methylamine, dimethylamine, ethylamine, diethylamine, propylamine and n-butylamine exists in the bulk drug or the finished drug.

The invention has the following beneficial effects:

aiming at the situation that short-chain fatty amine impurities are easy to remain in the preparation process of the traditional bulk drug and the like and have potential safety hazards, the invention provides a method for simultaneously detecting various short-chain fatty amines. The method can detect the effective components and various short chain fatty amines in the bulk drug and the like simultaneously under the same chromatographic condition, has good separation effect, high sensitivity, good solution stability and specificity meeting the requirements, can better control the product quality of the bulk drug or the finished drug, and improves the safety.

Drawings

FIG. 1 is a liquid chromatogram of a blank solution in a specificity study of example 2 of the present invention.

FIG. 2 is a liquid chromatogram of an ammonia control solution in a specificity study of example 2 of the present invention.

FIG. 3 is a liquid chromatogram of a methylamine control solution in the specificity study of example 2 of the invention.

FIG. 4 is a liquid chromatogram of a dimethylamine control solution in the specificity study of example 2 of the present invention.

FIG. 5 is a liquid chromatogram of a mixed control solution in a specificity study of example 2 of the present invention.

FIG. 6 is a liquid chromatogram of a labeled test solution for use in a specificity study according to example 2 of the present invention.

FIG. 7 is a liquid chromatogram of the sample solution labeled in example 3 of the present invention.

FIG. 8 is a liquid chromatogram of the sample solution labeled in example 6 of the present invention.

FIG. 9 is a gas chromatogram of the sample solution labeled in comparative example 1 of the present invention.

FIG. 10 is a liquid chromatogram of the test sample solution labeled in comparative example 2 according to the present invention.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Vonolamine fumarate bulk drug: hunan Qianjun Xiangjiang pharmaceutical Co., ltd., lot numbers 190302, 190401, 190402.

Control:

ammonia: ACS, lot 82C1908PG, content about 25%;

methylamine: the national drug group, lot number 20181113, content about 33%;

dimethylamine: shanghai Ala latin, lot F2012030, content about 40%.

Reagent solvent:

4-fluoro-3-nitrobenzotrifluoride: shanghai Miclin Biochemical technologies Co., ltd., lot C13763497;

dimethylformamide: kangkode, lot number 210224;

n-diisopropylethylamine: shanghai Miclin Biochemical technologies Co., ltd., lot number M180830684.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1 method for simultaneously detecting ammonia, methylamine and dimethylamine in vonolamine fumarate bulk drug

The embodiment provides a method for simultaneously detecting ammonia, methylamine and dimethylamine in a vonolamine fumarate bulk drug, which adopts an HPLC method, and specifically comprises the following steps:

1. solution preparation

1-1, sample preparation solvent: dimethylformamide (hereinafter abbreviated as DMF).

1-2, derivatization solution: about 1g of 4-fluoro-3-nitrobenzotrifluoride (FNBT for short later) is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by DMF, and uniformly mixed to obtain the finished product.

1-3, catalyst solution: 1ml of 1% N, N-diisopropylethylamine (hereinafter abbreviated as DIPEA) solution is dissolved in 100ml of DMF and evenly mixed to obtain the catalyst.

1-4, blank solution: taking 0.5ml of DMF, adding 400 μl of derivatization solution and 150 μl of catalyst solution, mixing, heating in water bath at 80deg.C for 90min, and cooling.

1-5, test sample solution: taking about 12mg of the vonolamine fumarate bulk drug, precisely weighing, placing into a 50ml measuring flask, dissolving with DMF, diluting to a scale, and shaking uniformly; precisely weighing 0.5ml of the solution, placing into a sample injection vial, respectively adding 400 μl of the derivatization solution and 150 μl of the catalyst solution, mixing, heating in water bath at 80deg.C for 90min, and cooling.

1-6, ammonia control solution: taking about 10mg of ammonia water, precisely weighing, placing into a 50ml measuring flask, dissolving with DMF, diluting to a scale, and shaking uniformly to obtain stock solution; precisely measuring 10 μl of stock solution, placing into a sample injection vial, adding 600 μl of derivatization solution and 300 μl of catalyst solution respectively, mixing, heating in water bath at 80deg.C for 90min, and cooling.

1-7, methylamine reference solution: taking about 10mg of methylamine solution, precisely weighing, placing into a 50ml measuring flask, dissolving with DMF, diluting to scale, and shaking uniformly to obtain stock solution; precisely measuring 10 μl of stock solution, placing into a sample injection vial, adding 600 μl of derivatization solution and 300 μl of catalyst solution respectively, mixing, heating in water bath at 80deg.C for 90min, and cooling.

1-8, dimethylamine control solution: about 10mg of dimethylamine solution is taken, precisely weighed, placed in a 50ml measuring flask, dissolved and diluted to the scale by DMF, and shaken uniformly to be used as stock solution; precisely measuring 10 μl of stock solution, placing into a sample injection vial, adding 600 μl of derivatization solution and 300 μl of catalyst solution respectively, mixing, heating in water bath at 80deg.C for 90min, and cooling.

1-9, mixing control stock solution: respectively weighing about 50mg, 42mg and 31mg of ammonia water, methylamine solution and dimethylamine solution respectively, placing into a same 50ml measuring flask, dissolving with DMF, diluting to scale, and shaking uniformly to obtain the final product.

1-10, mixing reference substance mother liquor: precisely measuring 0.25ml of mixed reference stock solution, placing into a 50ml measuring flask, diluting to scale with DMF, and shaking.

1-11, mixing the reference substance solution: precisely measuring 0.5ml of mother liquor of the mixed reference substance, placing into a sample injection small bottle, respectively adding 400 μl of derivatization solution and 150 μl of catalyst solution, mixing, heating in water bath at 80deg.C for 90min, and cooling.

1-12, adding a labeled test sample solution: taking about 12mg of voronoi fumarate bulk drug, precisely weighing, placing in a 50ml measuring flask, precisely measuring 0.25ml of reference stock solution, placing in the same 50ml measuring flask, adding DMF to dissolve and dilute to scale, shaking uniformly, precisely measuring 0.5ml, placing in a sample injection vial, respectively adding 400 μl of derivatization solvent and 150 μl of catalyst, mixing uniformly, taking out after water bath at 80deg.C for 90min, and cooling.

2. Conditions of liquid chromatography

Chromatographic column: octadecylsilane chemically bonded silica was used as a filler, specifically a column Phenomenex Ultremex u C18, 4.6mm.times.250 mm,5 μm.

The detection wavelength is 416nm, the column temperature is 45 ℃, the flow rate is 1.0ml per minute, and the sample injection volume is 5 μl;

mobile phase a:0.1% formic acid in water; mobile phase B: methanol: acetonitrile (v/v, 40:60), elution was performed with a linear gradient according to Table 1.

TABLE 1 gradient elution parameters

Time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0	38	62
3	38	62
			8	20	80
16	20	80
			17	38	62
23	38	62

3. Detection and results

Taking 5 mu L of mixed reference substance solution and 5 mu L of sample solution to be tested for sample injection detection, recording peak areas of ammonia, methylamine and dimethylamine in a chromatogram, and calculating the content of impurities of the ammonia, methylamine and dimethylamine in the sample according to an external standard method:

wherein S is _Impurity(s) : the area of the impurity peak in the sample solution;

S _{for a pair of} : the area of the corresponding impurity peak in the mixed reference substance solution;

M _{for a pair of} : weighing the reference substance, and mg;

M _sample : weighing the test sample, and mg;

ω _{for a pair of} : control content,%.

As shown in table 2, it was found that the three batches of vonolamine fumarate crude drugs all had methylamine impurities, and that the residues were 0.01 to 0.02%.

TABLE 2 detection results of impurities of ammonia, methylamine and dimethylamine in vonolamine fumarate bulk drug

EXAMPLE 2 methodology investigation

Methodology study verification was performed on the method of example 1, the verification project being specifically as follows:

1. specialization of

The blank solution, the ammonia reference solution, the methylamine reference solution, the dimethylamine reference solution, the mixed reference solution and the standard sample solution are respectively measured and detected by sample injection according to the liquid chromatography conditions of the embodiment 1, the chromatograms of the blank solution, the mixed reference solution and the standard sample solution are respectively shown in fig. 1-6, and the detection results are shown in table 3.

TABLE 3 example 2 solution separation results for labeled test

Name of the name	Retention time/min	Degree of separation
			Ammonia	7.444	/
Methylamine	10.070	11.69
			Dimethylamine	10.928	3.74

The result shows that the retention time of the main component, namely the voronoi fumarate, is about 21 minutes, and the blank solution has a dimethylformamide solvent (about 8 minutes of peak) in the chromatogram, so that the blank solution is free of interference; under the chromatographic condition, the peak time of each impurity in the labeled sample is far earlier than that of the main component, the separation degree between adjacent impurities is more than 1.5, the measurement of the main component, namely, the vonolamine fumarate, is not interfered, the impurities do not interfere the main component peak, the main component peak purity of the labeled sample solution also meets the requirement, and the specificity of the method is good.

2. Detection Limit (LOD) and quantification Limit (LOQ) assays

The mixed control solution was diluted stepwise and tested using the liquid chromatography conditions of example 1, the results are shown in table 4.

TABLE 4 limit of detection and limit of quantification results

The result shows that the method has lower detection limit and quantitative limit for 3 amine impurities and better sensitivity.

3. Solution stability investigation

To examine the stability of the solutions in the method of the present invention, a mixed control solution was prepared according to example 1, and placed at room temperature for 0h, 4h, 8h, 12h, and 24h, respectively, and injected into a liquid chromatograph, and detected according to the liquid chromatograph conditions of example 1, and a chromatogram was recorded, and the relative change rates of the peak areas of the impurities at different times as compared with the peak area of 0 hour were measured and calculated, and the results are shown in table 5.

TABLE 5 solution stability results

The results show that after the control solution is placed at room temperature for 24 hours, the area change rate of the peaks of 3 amine impurities is less than 10%, which indicates that the solution is stable within 24 hours after being placed at room temperature.

4. Durability inspection

(1) To analyze the effect of the volumes of derivatization solutions on derivatization, labeled test solutions were prepared from different derivatization solution volumes, and the prepared test solutions were tested according to the liquid chromatography conditions of example 1, with the results shown in table 6 below.

TABLE 6 influence of derivatization solution volumes on derivatization

The results showed that there was no significant difference in the volumes of the derivatization solution between 400 μl and 500 μl, and that of ammonia, methylamine, dimethylamine, S V (peak area volume), so the derivatization solution was determined to be 400 μl. The amount of the derivatization solution added is related to the content of the impurities (i.e., ammonia, methylamine, dimethylamine) contained, and the impurities contained need to be completely reacted for derivatization.

(2) To analyze the effect of the water bath time on derivatization, a mixed control solution and a labeled test solution were prepared with different water bath times, and the prepared mixed control solution and labeled test solution were tested, and the results are shown in table 7 below.

TABLE 7 influence of Water bath time on derivatization

The result shows that the mixed reference substance solution is derived for 45-120 min, the peak areas of the solvents are not obviously different, the standard sample solution is added for 45-120 min, the peak areas of the solvents are not obviously different, and the water bath time is preferably 90min.

(3) In order to examine whether or not the accuracy of the impurity measurement result is affected and the degree of the influence when the chromatographic parameter in the chromatographic condition slightly fluctuates, the chromatographic condition of example 1 was examined: the single factor of the chromatographic conditions of example 1 was changed in accordance with Table 8, and the results of the measurement were shown in Table 9.

Table 8 durability liquid chromatography single factor variable settings

TABLE 9 results of determination of durability liquid chromatography single factor variable

Note that: the minimum degree of separation is the degree of separation between methylamine and dimethylamine.

The results show that the measurement results under different single factor variable conditions are extremely poor and less than 0.02%, and the results show that no obvious difference exists, and the chromatographic conditions can be detected.

Example 3 method for simultaneously detecting Ammonia and dimethylamine in methylamine solution

The embodiment provides a method for simultaneously detecting ammonia and dimethylamine in a methylamine solution, and the specific implementation mode refers to the embodiment 1 for detection, which is different in that the preparation of a reference substance solution and a test sample solution is as follows:

3-1, test sample solution: precisely weighing 50 μl of methylamine solution, precisely weighing, placing into a 50ml measuring flask, adding appropriate amount of DMF to dissolve, adding 5ml of derivatization solution and 1ml of catalyst solution, mixing, standing in water bath at 80deg.C for 90min, taking out, cooling, adding DMF to scale, and shaking.

3-2, mixing reference substance mother liquor: precisely measuring 50 μl of ammonia water and dimethylamine solution, precisely weighing, placing into the same 50ml measuring flask, dissolving with DMF, diluting to scale, and shaking.

3-3, mixing the reference substance solution: precisely measuring 0.25ml of mixed reference substance mother liquor, placing into a 50ml measuring flask, adding appropriate amount of DMF to dissolve, adding 5ml of derivatization solution and 1ml of catalyst solution, mixing, standing in water bath at 80deg.C for 90min, taking out, cooling, diluting to scale with DMF, and shaking.

3-4, adding a labeled test sample solution: taking 50 mu l of methylamine alcohol solution, precisely weighing, placing into a 50ml measuring flask, adding 250 mu l of mixed reference substance mother liquor, placing into the same 50ml measuring flask, adding a proper amount of DMF to dissolve, adding 5ml of derivatization solution and 1ml of catalyst solution, uniformly mixing, placing in a water bath at 80 ℃ for 90min, taking out, cooling, dissolving with DMF, diluting to a scale, and shaking uniformly to obtain the final product.

The results of the detection are shown in tables 10 to 11 and FIG. 7.

TABLE 10 example 3 solution separation results for labeled test sample

TABLE 11 EXAMPLE 3 addition of the labeled sample solution ammonia, dimethylamine content

The results show that the separation degree between the methylamine solution and 2 amine impurities meets the requirement, and ammonia and dimethylamine impurities are detected in 2 batches of methylamine.

Example 4 method for simultaneously detecting Ammonia and dimethylamine in methylamine solution

This example provides a method for simultaneously detecting ammonia and dimethylamine in a methylamine solution, and the specific embodiment refers to example 3, except that the addition amount of the derivatization solution is 4.8-5.2 ml when the standard sample solution is added for preparation, and the results are shown in table 12.

Table 12 example 4 detection results of labeled test solutions

Derivatization solution addition/ml	Ammonia content/%	Dimethylamine content/%
			5.0	0.20	0.14
4.8	0.18	0.12
			5.2	0.17	0.12
Extremely poor	0.03	0.02

The results showed that the addition of the derivatization solution varied by + -0.2 ml, and that there was no significant difference in ammonia and dimethylamine levels.

Example 5A method for simultaneously detecting Ammonia and dimethylamine in a methylamine solution

This example provides a method for simultaneously detecting ammonia and dimethylamine in a methylamine solution, and the specific embodiment refers to example 3 for detection, except that the water bath time is 75-105 min when the labeled sample solution is prepared, and the results are shown in table 13.

TABLE 13 example 5 detection results of labeled test solutions

The results show that the addition amount of the derivatization solution changes by +/-15 min, and the ammonia content and the dimethylamine content have no obvious difference.

Example 6 method for simultaneously detecting Ammonia and dimethylamine in methylamine solution

This example provides a method for simultaneously detecting ammonia and dimethylamine in a methylamine solution, and the specific embodiment refers to example 3 for detection, wherein mobile phase B is acetonitrile and methanol respectively: acetonitrile (v/v, 40:60), results are shown in Table 14 and FIG. 8.

TABLE 14 example 6 detection results of labeled test solutions

Mobile phase B	Degree of separation of ammonia from methylamine	Degree of separation of methylamine from dimethylamine
			Acetonitrile	9.41	Without any means for
Methanol: acetonitrile (40:60)	9.85	2.83
			Methanol: acetonitrile (30:70)	12.39	3.89

The results show that when the mobile phase B contains no methanol, the methylamine and dimethylamine cannot be separated; the methanol content is between 30% and 40%, the separation degree is more than 1.5, and the requirements are met.

Comparative example 1 method for detecting ammonia, methylamine and dimethylamine in vonolamine fumarate bulk drug

The method for detecting ammonia, methylamine and dimethylamine in the vonolamine fumarate bulk drug adopts a gas chromatography method, and the solution preparation and gas chromatography conditions are as follows:

adding a labeled test sample solution: taking about 0.8g of the vonolamine fumarate bulk drug, precisely weighing, placing into a 10ml measuring flask, adding a proper amount of dimethylformamide, carrying out ultrasonic treatment to dissolve, adding 50 mu l of methanol and acetonitrile respectively, adding 10 mu l of methylamine and dimethylamine respectively, diluting to a scale with dimethylformamide, and shaking uniformly to obtain the final product.

Gas chromatography conditions: using a CP-Volamine capillary column (60 m 0.32mm 0.25 μm) as a chromatographic column; the initial temperature was 35℃for 5 minutes, and the temperature was raised to 200℃at a rate of 10℃per minute for 5 minutes; the detector is a hydrogen flame ionization detector, the carrier gas is nitrogen, and the temperature of the detector is 250 ℃; the temperature of the sample inlet is 150 ℃; the sample volume was 1. Mu.l.

The labeled sample solution is subjected to sample injection detection according to the gas chromatography conditions, and the result is shown in fig. 9. As can be seen, ammonia does not respond, does not peak, and dimethylamine and methylamine are both poorly separated and have poor peaks.

Comparative example 2 method for detecting Ammonia and dimethylamine in methylamine solution

This comparative example provides a method for detecting ammonia and dimethylamine in a methylamine solution, and the specific embodiment is performed with reference to example 3, except that mobile phase a is a 0.1% phosphoric acid aqueous solution, and the result is shown in fig. 10. It can be seen that methylamine and dimethylamine cannot be separated.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting a plurality of short-chain aliphatic amines is characterized in that a sample to be detected is firstly treated by a catalyst organic amine compound and a derivatization agent halogenated nitrobenzene compound, and then is detected according to the following high performance liquid chromatography conditions:

chromatographic column: octadecylsilane chemically bonded silica packed column, detection wavelength: 400-430 nm, column temperature: the temperature is 35-50 ℃ and the flow rate is 0.9-1.1 ml/min; mobile phase a: 0.05-0.15% formic acid aqueous solution, mobile phase B: methanol and acetonitrile (25-40): the mixed solution of (60-75) was tested under the following gradient elution conditions:

。

2. the method for simultaneously detecting a plurality of short-chain aliphatic amines according to claim 1, wherein the treatment temperature of the catalyst organic amine compound and the derivatizing agent halogenated nitrobenzene compound is 55-85 ℃.

3. The method for simultaneously detecting a plurality of short-chain aliphatic amines according to claim 1, wherein the catalyst organic amine compound and the derivatizing agent halogenated nitrobenzene compound are treated for 45-120 min.

4. The method for simultaneously detecting a plurality of short-chain aliphatic amines according to claim 1, wherein the derivatizing agent is halogenated nitrobenzene compound comprising 4-fluoro-3-nitrobenzotrifluoride, 1-fluoro-2-nitrobenzene, 1-fluoro-4-nitrobenzene or 2, 4-dinitrobenzene.

5. The method for simultaneously detecting a plurality of short-chain aliphatic amines according to claim 1, wherein the catalyst organic amine compound comprises N, N-diisopropylethylamine or triethylamine.

6. The method for simultaneous detection of multiple short-chain fatty amines according to claim 1, wherein the chromatographic column is Phenomenex Ultremex u c18,4.6mm x 250mm,5 μm.

7. The method for simultaneous detection of multiple short-chain fatty amines according to claim 1, wherein the short-chain fatty amine comprises ammonia, methylamine, dimethylamine, ethylamine, diethylamine, propylamine, or n-butylamine.

8. The method for simultaneously detecting a plurality of short-chain aliphatic amines according to claim 7, wherein the ammonia has a detection limit of 0.002%, the methylamine has a detection limit of 0.003%, and the dimethylamine has a detection limit of 0.01%.

9. The method for simultaneously detecting a plurality of short-chain aliphatic amines according to claim 7, wherein the quantitative limit of ammonia or methylamine is 0.01%, and the quantitative limit of dimethylamine is 0.03%.

10. Use of the method for simultaneously detecting a plurality of short-chain fatty amines according to any of claims 1 to 9 in quality monitoring of bulk drugs or finished drugs.