CN108037221B

CN108037221B - Method for simultaneously separating and determining methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by liquid chromatography

Info

Publication number: CN108037221B
Application number: CN201810101645.2A
Authority: CN
Inventors: 李莉; 张明; 顾浦中; 马炳原; 崔红艳; 李宗豪; 钟家棋; 沈梦元; 蒋荷
Original assignee: Kangda College Of Nanjing Medical University
Current assignee: Kangda College Of Nanjing Medical University
Priority date: 2018-02-01
Filing date: 2018-02-01
Publication date: 2020-11-17
Anticipated expiration: 2038-02-01
Also published as: CN108037221A

Abstract

The invention discloses a method for simultaneously separating and measuring methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by using a liquid chromatography, which comprises the steps of measuring a sample solution in a high performance liquid chromatograph, and calculating the contents of the methionine sulfoxide and methionine sulfone impurities in the sample by peak area according to a standard curve method. The detection method is simple, convenient and feasible, short in analysis time, strong in specificity and good in reproducibility, and can effectively determine and separate methionine sulfoxide and methionine sulfone impurities in the compound amino acid injection 18 AA.

Description

Method for simultaneously separating and determining methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by liquid chromatography

Technical Field

The invention belongs to the technical field of a medicine quality determination method, and particularly relates to a method for separating and detecting methionine degradation impurities methionine sulfoxide and methionine sulfone in compound amino acid injection 18AA by using a high performance liquid chromatograph.

Background

The compound amino acid injection 18AA is mainly used for patients who have insufficient protein intake, malabsorption and the like and have amino acid which can not meet the metabolic needs of organisms, and is also used for improving the nutritional status of patients after operations. The rapid development of the compound amino acid injection ensures that clinical critically ill patients obtain good parenteral nutrition support, ensures the positive nitrogen balance of organisms and promotes the synthesis of protein.

With the growing public and media concern over the safety of pharmaceutical products, impurities in pharmaceutical products have become important indicators in the quality control of pharmaceutical products. In recent years, with the advent and popularization of new analysis and detection technologies, trace amounts of harmful substances in medicines are gradually clarified to reveal chemical structures or material bases, and some rapid screening platforms emerge on the safety research of impurities, so that a foundation is laid for quality control of products in the future. The correlation between impurity levels and drug safety is well studied, and this is one of the directions for future research.

The work of assessing the purity of amino acids has been a challenge due to the lack of chromophores. In the european pharmacopoeia in the past, impurity monitoring of amino acid starting materials has generally been by means of thin layer chromatography with ninhydrin reagent developed color, however, the sensitivity and performance of this method is poor. The impurity examination of amino acids in the new Edition of European Pharmacopoeia was changed to the analytical method for post-ion-exchange-column ninhydrin derivatization (EDQM, European Pharmacopoeia 8th Edition, 2015).

Methionine sulfoxide is an oxidation impurity of methionine, and has a certain toxic injury effect on male liver cells. Methionine sulfone is also an oxidizing impurity of methionine, and can inhibit the biosynthesis of glutamic acid.

The existence of two impurities, namely methionine sulfoxide and methionine sulfone, poses a potential threat to the clinical medication safety of the compound amino acid injection 18 AA. However, no relevant report is found on a detection method for simultaneously separating and detecting methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA through literature search.

Therefore, a method for scientifically and effectively separating and determining methionine sulfoxide and methionine sulfone impurities in the compound amino acid injection 18AA simultaneously is needed, so that the detection cost can be reduced, the labor intensity can be reduced, and the method is energy-saving and environment-friendly.

Disclosure of Invention

The invention aims to provide a method for simultaneously separating and determining methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by using a liquid chromatography.

In order to achieve the above object, the present invention employs the following:

a method for separating and determining methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA simultaneously by liquid chromatography comprises determining a sample solution in a high performance liquid chromatograph, and calculating the contents of methionine sulfoxide and methionine sulfone impurities in the sample by peak area according to a standard curve method;

the chromatographic conditions are as follows:

the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column;

the mobile phase A is 0.01 mol.L^-1Diammonium hydrogen phosphate solution containing 0.01 mol.L^-1Adjusting the pH value of the octane sodium sulfonate buffer solution to 1.9 +/-0.05 by using phosphoric acid;

the mobile phase B is acetonitrile;

mobile phase elution procedure:

time/min	Mobile phase A/%)	Mobile phase B/%)
			0	100	0
22	100	0
			23	90	10
29	90	10
			30	100	0
35	100	0

The flow rate was 1.0ml per minute;

the detection wavelength is 205 nm;

the column temperature is 4 ℃;

the injection volume was 100. mu.l.

Further, the chromatographic column is a Titank C18 chromatographic column; the Titank C18 chromatographic column has a specification of 4.6mm × 250mm, 5 μm.

Further, in the method, the sample solution is: taking a methionine sulfoxide reference substance, precisely weighing, dissolving with water, and quantitatively diluting to obtain a reference substance solution containing about 1mg of methionine sulfoxide per 1ml, as methionine sulfoxide reference substance stock solution; taking a methionine sulfone reference substance, precisely weighing, dissolving with water, and quantitatively diluting to obtain a reference substance solution containing about 1mg of methionine sulfone per 1ml, as a methionine sulfone reference substance stock solution; precisely measuring 1ml of each of the compound amino acid injection 18AA, the methionine sulfoxide reference substance storage solution and the methionine sulfone reference substance storage solution, mixing, placing in a 10ml measuring flask, adding water to dilute to scale, and shaking up to obtain a test solution.

Further, in the method, a control solution is prepared, a high performance liquid chromatograph is used for detecting the control solution, linear regression is carried out by taking the peak area as a vertical coordinate and the sample concentration as a horizontal coordinate, a standard curve is drawn, and a regression equation is obtained; the preparation control solutions were: precisely measuring a proper amount of methionine sulfoxide reference stock solution, diluting with water, and respectively preparing solutions containing about 0.30, 5, 10, 50, 100 and 200 μ g of methionine sulfoxide per 1ml as methionine sulfoxide reference solutions; precisely measuring appropriate amount of methionine sulfone control stock solution, diluting with water, and making into solution containing methionine sulfone 4.20, 5, 10, 50, 100 and 200 μ g per 1ml as methionine sulfone control solution.

The invention has the following advantages:

the detection method is simple, convenient and feasible, has short analysis time, strong specificity and good reproducibility, can simultaneously separate and detect the methionine sulfoxide and methionine sulfone impurities in the compound amino acid injection 18AA within 35 minutes, and provides the separation and determination problem which cannot be solved by the prior art, thereby ensuring the controllable quality of the compound amino acid injection 18AA and finally determining the safety and effectiveness of the product.

Drawings

FIG. 1 is a chromatogram of compound amino acid injection 18 AA.

FIG. 2 is a chromatogram of a test solution.

FIG. 3 is a chromatogram of the methionine sulfoxide linear control solution (5).

FIG. 4 is a chromatogram of the methionine sulfone linear control solution (5).

FIG. 5 is a linear graph of methionine sulfoxide.

FIG. 6 is a linear plot of methionine sulfone.

FIG. 7 is a chromatogram of a test solution under normal conditions.

FIG. 8 is a chromatogram of a test solution at flow rate + 10%.

FIG. 9 is a chromatogram of a test solution at a flow rate of-10%.

FIG. 10 is a chromatogram of a test solution at an organic phase ratio of + 1%.

FIG. 11 is a chromatogram of a test solution under the condition of organic phase comparative example-1%.

FIG. 12 is a chromatogram of a test solution at mobile phase pH + 0.05.

FIG. 13 is a chromatogram of a sample solution under a condition of mobile phase pH-0.05.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Reagents, drugs, instruments and equipment used in the embodiments of the present invention are known products, and can be obtained by purchasing commercially available products. The method comprises the following specific steps:

reagents and drugs:

methionine sulfoxide control (arkpharminc, batch # WG 0002486-160825001); methionine sulfone control (Beijing Bailingwei science and technology Co., Ltd., batch No.: LCC0Q 84);

compound amino acid injection (18AA) (Chenxin pharmaceutical Co., Ltd., batch No. 1611052143);

acetonitrile (TEDIA, batch: AS1122-801) AS chromatographically pure;

diammonium phosphate (national medicine group chemical reagent Co., Ltd., batch No. 20170301)

Sodium octane sulfonate (Saen chemical technology Shanghai Co., Ltd., batch No.: EI050108) was analytically pure.

The instrument equipment comprises:

waters1525 hplc;

shanghai thunder magnetic table type precise digital display PHS-3G acidimeter;

one-tenth ten-thousandth type of precision electronic balance: AUW 220D;

the model of the circulating water vacuum pump is as follows: SHB-III.

Example 1

A method for simultaneously separating and determining methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by using liquid chromatography comprises the following steps:

1) preparation of control stock solutions:

taking a methionine sulfoxide reference substance, precisely weighing, dissolving with water, and quantitatively diluting to obtain a reference substance solution containing about 1mg of methionine sulfoxide per 1ml, as methionine sulfoxide reference substance stock solution.

Taking a methionine sulfone control, precisely weighing, dissolving with water, and quantitatively diluting to obtain a control solution containing about 1mg of methionine sulfone per 1ml, as a methionine sulfone control stock solution.

2) Preparation of control solutions

Precisely measuring appropriate amount of methionine sulfoxide control stock solution, diluting with water, and making into solution containing methionine sulfoxide 0.30, 5, 10, 50, 100 and 200 μ g per 1ml as methionine sulfoxide control solutions (1), (2), (3), (4), (5) and (6).

Precisely measuring appropriate amount of methionine sulfone control stock solution, diluting with water, and making into solution containing methionine sulfone 4.20, 5, 10, 50, 100 and 200 μ g per 1ml as methionine sulfone control solutions (1), (2), (3), (4), (5) and (6).

3) Preparing test solution

Because the content of two impurities, namely methionine sulfoxide and methionine sulfone, in the compound amino acid injection 18AA sample is very small, the exploration of chromatographic conditions is not convenient, and in order to examine the chromatographic conditions, a test solution is prepared in the following way:

precisely measuring 1ml of each of the compound amino acid injection 18AA, the methionine sulfoxide reference substance storage solution and the methionine sulfone reference substance storage solution, mixing, placing in a 10ml measuring flask, adding water to dilute to scale, and shaking up to obtain a test solution.

4) Measurement of

Respectively taking 100 mu l of each of the methionine sulfoxide linear control solutions (1) - (6), the methionine sulfone linear control solutions (1) - (6) and the test solution, injecting into a high performance liquid chromatograph, and recording chromatograms. Typical maps are shown in FIGS. 1-4.

The chromatographic conditions are as follows:

a chromatographic column: titank C18, 4.6mm × 250mm, 5 μm;

mobile phase B: acetonitrile;

linear gradient elution was performed as per Table 1 at a flow rate of 1.0ml per minute;

detection wavelength: 205 nm;

column temperature: 4 ℃;

sample introduction volume: 100 μ l.

TABLE 1 gradient elution procedure

In FIG. 2, the retention time is 14.780 minutes for methionine sulfoxide and 16.465 minutes for methionine sulfone. The separation degree between methionine sulfone and methionine sulfoxide is 1.8, and methionine sulfone and adjacent peak, methionine sulfoxide and adjacent peak are well separated, and fig. 2 illustrates that under the condition, the invention purpose of the invention can be realized.

Example 2 (Linear Range)

Methionine sulfoxide control solutions (1), (2), (3), (4), (5) and (6) and methionine sulfone control solutions (1), (2), (3), (4), (5) and (6) were prepared according to the preparation methods of the methionine sulfoxide control solution and the methionine sulfone control solution in example 1, respectively.

Precisely measuring 100 μ l of each of methionine sulfoxide control solutions (1), (2), (3), (4), (5) and (6) and methionine sulfone control solutions (1), (2), (3), (4), (5) and (6), respectively injecting into a high performance liquid chromatograph, recording chromatogram, and measuring peak area. Taking the peak area A as an ordinate and the sample concentration C as an abscissa, performing linear regression, and calculating a correlation coefficient (r).

TABLE 2 methionine sulfoxide linearity and Range test results

TABLE 3 methionine sulfone Linear and Range test results

As a result, methionine sulfoxide content was 0.31 to 201.76. mu.g/ml^-1The linear curve of (a) is 44217C-3774, and r is 1 (see table 2, fig. 5 for details). Methionine sulfone content of 4.51-225.72 μ g/ml^-1The linear curve of (a) 2618C-1360, r 0.9999 (see table 3, fig. 6 for details). The sample injection concentrations of methionine sulfoxide and methionine sulfone have obvious linear relation with the corresponding peak area response values.

Example 3 (precision)

1. Sample introduction precision:

an appropriate amount of the methionine sulfoxide control stock solution was precisely measured, and diluted with water to prepare a solution containing about 10. mu.g of methionine sulfoxide per 1ml, and under the chromatographic conditions of example 1, sample introduction was performed continuously for 6 times, and the RSD of the peak area of methionine sulfoxide was 0.16% (Table 4).

An appropriate amount of the methionine sulfone control stock solution was precisely measured, and diluted with water to prepare a solution containing about 10. mu.g of methionine sulfone per 1ml, and under the chromatographic conditions of example 1, sample injection was performed continuously 6 times, and the RSD of the methionine sulfone peak area was 1.84% (Table 5). As is clear from tables 4 and 5, the reproducibility was good.

TABLE 4 methionine sulfoxide linearity and Range test results

TABLE 5 methionine sulfone Linear and Range test results

Sample introduction sequence	Peak area
			1	28458
2	29655
		3	29798
4	29329
		5	29288
6	28637
		Mean value of	29341.4
RSD(％)	1.84

2. And (3) repeatability test:

6 test solutions were prepared in parallel according to the method of example 1. Precisely measuring 6 parts of test solution, methionine sulfoxide control solutions (1), (2), (3), (4), (5) and (6) and methionine sulfone control solutions (1), (2), (3), (4), (5) and (6), respectively performing sample injection analysis, and determining that the average content of methionine sulfoxide is 1003.1 μ g/ml^-1RSD is 0.13%; the average content of methionine sulfone is 1182.4 mug ml^-1RSD was 1.41% (see Table 6 for details). The data result shows that the method has good repeatability.

TABLE 6 results of repeated experiments

Example 4 (solution stability experiment)

The sample solution is placed at room temperature, and is respectively measured after 0, 3, 6, 10, 14, 17, 20 and 24 hours, the sample injection volume is 100 mu l, and the stability of the sample solution is examined by using the main peak area and the impurity content of the sample solution. The results are shown in Table 7:

TABLE 7 test results of solution stability of test article

As is clear from Table 7, the test solution was stable for 24 hours at room temperature.

Example 5 (accuracy: recovery from sample application experiment)

Preparing a solution:

taking a methionine sulfoxide (MetsoX) reference substance, precisely weighing, dissolving with water, and quantitatively diluting to obtain a reference substance solution containing about 1mg of methionine sulfoxide per 1ml, wherein the reference substance solution is used as a methionine sulfoxide reference substance stock solution.

Taking a methionine sulfone (MetsoN) reference substance, precisely weighing, dissolving with water, and quantitatively diluting to obtain a reference substance solution containing about 1mg of methionine sulfone per 1ml, wherein the reference substance solution is used as a methionine sulfone reference substance stock solution.

Methionine sulfoxide control solutions (1), (2), (3), (4), (5) and (6) and methionine sulfone control solutions (1), (2), (3), (4), (5) and (6) were prepared according to the method of example 1.

Respectively adding 50 mu g/ml into compound amino acid injection (18AA)^-1、100μg·ml^-1、1000μg·ml^-1The methionine sulfoxide and the methionine sulfone (the specific preparation method is that 1ml of compound amino acid injection (18AA) is precisely measured and put into a 10ml measuring flask, and the concentration of the compound amino acid injection and the methionine sulfone are respectively added into the measuring flask with the concentration of about 50 mu g/ml^-1、100μg·ml^-1、1000μg·ml^-11ml each of the methionine sulfoxide and methionine sulfone solutions) of (1), 3 parts of the solutions were prepared in parallel for each concentration point, and the contents of the above-mentioned impurities were measured in accordance with the measurement method of example 1. And calculating the sample recovery rate and the RSD of each impurity. The recovery results are shown in Table 8.

Table 8 sample recovery test results (n ═ 9)

As can be seen from Table 8, the recovery rates of methionine sulfoxide (MetsoX) and methionine sulfone (MetsoN) in the method are respectively 99.09-102.14% and 98.20-101.21%, and the determination accuracy is good.

Example 6 (durability test)

The flow rate of the mobile phase, the pH of the mobile phase and the ratio of the organic phase were adjusted appropriately, the chromatographic conditions in example 1 were varied to a small extent, and the durability of the chromatographic conditions was examined by comparing the results of the measurement of methionine sulfoxide and methionine sulfone in the same sample under different conditions, as shown in table 9, and the chromatograms are shown in fig. 7 to 13.

FIG. 7 is a chromatogram of a test solution under normal conditions. FIG. 8 is a chromatogram of a test solution at flow rate + 10%. FIG. 9 is a chromatogram of a test solution at a flow rate of-10%. FIG. 10 is a chromatogram of a test solution at an organic phase ratio of + 1%. FIG. 11 is a chromatogram of a test solution under the condition of organic phase comparative example-1%. FIG. 12 is a chromatogram of a test solution at mobile phase pH + 0.05. FIG. 13 is a chromatogram of a sample solution under a condition of mobile phase pH-0.05.

TABLE 9 durability test results

Note: -^aThe chromatogram showed no degree of separation.

As can be seen from table 9, methionine sulfoxide (MetsoX) and methionine sulfone (MetsoN) were overlapped with adjacent peaks respectively without separation under the condition of the organic phase ratio + 1% with a small change in the flow rate of the mobile phase, the pH of the buffer salt, and the mobile phase composition ratio. Although the retention time of methionine sulfoxide (MetsoX) and methionine sulfone (MetsoN) in other conditions slightly varies, the measurement results are basically consistent, and the measurement durability of related substances is good. Therefore, the ratio of the organic phase must be strictly controlled within a predetermined range during the experiment.

The detection method uses an acidic ion pair reagent and an organic solvent as mobile phases, adopts high performance liquid chromatography to separate and measure methionine sulfoxide and methionine sulfone impurities in the compound amino acid injection 18AA, can separate and detect the methionine sulfoxide and methionine sulfone impurities simultaneously within 35 minutes, and has great significance for the research on the quality control and the amino acid degradation path of the compound amino acid injection 18AA medicine.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

Claims

1. A method for simultaneously separating and determining methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by using a liquid chromatography is characterized in that the method comprises the steps of firstly determining a sample solution in a high performance liquid chromatograph, and then calculating the contents of the methionine sulfoxide and methionine sulfone impurities in the sample by peak area according to a standard curve method;

the chromatographic conditions are as follows:

the mobile phase B is acetonitrile;

mobile phase elution procedure:

The flow rate was 1.0ml per minute;

the detection wavelength is 205 nm;

the column temperature is 4 ℃;

the injection volume was 100. mu.l.

2. The method of claim 1, wherein the chromatography column is a titank c18 chromatography column.

3. The method of claim 2, wherein the Titank C18 chromatography column has a size of 4.6mm x 250mm, 5 μm.

4. The method of claim 1, wherein the sample solution is: taking a methionine sulfoxide reference substance, precisely weighing, dissolving with water, and quantitatively diluting to obtain a reference substance solution containing about 1mg of methionine sulfoxide per 1ml, as methionine sulfoxide reference substance stock solution; taking a methionine sulfone reference substance, precisely weighing, dissolving with water, and quantitatively diluting to obtain a reference substance solution containing about 1mg of methionine sulfone per 1ml, as a methionine sulfone reference substance stock solution; precisely measuring 1ml of each of the compound amino acid injection 18AA, the methionine sulfoxide reference substance storage solution and the methionine sulfone reference substance storage solution, mixing, placing in a 10ml measuring flask, adding water to dilute to scale, and shaking up to obtain a test solution.

5. The method according to claim 1, wherein the method comprises preparing a control solution, detecting the control solution by using a high performance liquid chromatograph, performing linear regression with peak area as ordinate and sample concentration as abscissa, drawing a standard curve and obtaining a regression equation; the preparation control solutions were: precisely measuring a proper amount of methionine sulfoxide reference stock solution, diluting with water, and respectively preparing solutions containing about 0.30, 5, 10, 50, 100 and 200 μ g of methionine sulfoxide per 1ml as methionine sulfoxide reference solutions; precisely measuring appropriate amount of methionine sulfone control stock solution, diluting with water, and making into solution containing methionine sulfone 4.20, 5, 10, 50, 100 and 200 μ g per 1ml as methionine sulfone control solution.