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

Abstract

The invention discloses a method for simultaneous separation and determination of methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by liquid chromatography. According to the standard curve method, the content of methionine sulfoxide and methionine sulfone impurities in the test sample was calculated by peak area. The detection method of the invention is simple and feasible, has short analysis time, strong specificity and good reproducibility, and can effectively measure and separate the methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA.

Description

Simultaneous Separation and Determination of Methionine in Compound Amino Acid Injection 18AA by Liquid Chromatography Method for Sulfoxide and Methionine Sulfone Impurities

technical field

The invention belongs to the technical field of drug quality determination methods, in particular to a method for separating and detecting methionine in compound amino acid injection 18AA to degrade impurities of methionine sulfoxide and methionine sulfone by high performance liquid chromatography.

Background technique

Compound Amino Acid Injection 18AA is mainly used for patients with insufficient protein intake, malabsorption and other amino acids that cannot meet the metabolic needs of the body. It is also used to improve the nutritional status of patients after surgery. The rapid development of compound amino acid injection enables clinical critically ill patients to receive good parenteral nutrition support, ensures the body's positive nitrogen balance, and promotes protein synthesis.

With the increasing public and media attention to drug safety, impurities in drugs have become an important indicator in drug quality control. In recent years, with the advent and promotion of new analysis and detection technologies, trace amounts of harmful substances in medicines are gradually being elucidated to reveal their chemical structures or material basis, and some rapid screening platforms have also emerged in the safety research of impurities. The quality control of the product lays the foundation. Thorough research on the relationship between impurity levels and drug safety is also one of the directions for future research.

Due to the lack of chromophores, the purity assessment of amino acids has been difficult. In the past European Pharmacopoeia, the impurity monitoring of amino acid raw materials usually resorted to thin-layer chromatography with ninhydrin reagent color development, however, the sensitivity and performance of this method were poor. In the new version of the European Pharmacopoeia, the impurity test of amino acids was changed to the analytical method of ninhydrin derivatization after ion exchange column (EDQM, European Pharmacopoeia 8th Edition, 2015).

Methionine sulfoxide is an oxidized impurity of methionine, which has certain toxic and damaging effects on male hepatocytes. Methionine sulfone is also an oxidized impurity of methionine and can inhibit glutamate biosynthesis.

The existence of two impurities, methionine sulfoxide and methionine sulfone, poses a potential threat to the clinical safety of compound amino acid injection 18AA. However, through literature search, there is no relevant report on the simultaneous separation and detection of methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA.

Therefore, a scientific and effective method for simultaneous separation and determination of methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA is needed, which can not only reduce the detection cost, reduce labor intensity, but also save energy and protect the environment.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for simultaneously separating and measuring methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by liquid chromatography.

To achieve the above object, the present invention adopts the following content:

A method for simultaneously separating and measuring methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA by liquid chromatography. Calculate the content of methionine sulfoxide and methionine sulfone impurities in the test sample by peak area;

The chromatographic conditions are:

The chromatographic column is an octadecylsilane-bonded silica gel chromatographic column;

Mobile phase A is 0.01mol·L ^-1 diammonium hydrogen phosphate solution, which contains 0.01mol·L ^-1 sodium octane sulfonate buffer solution, and the pH value is adjusted to 1.9±0.05 with phosphoric acid;

Mobile phase B is acetonitrile;

Mobile phase elution procedure:

time/min Mobile phase A/% Mobile phase B/% 0 100 0 twenty two 100 0 twenty three 90 10 29 90 10 30 100 0 35 100 0

The flow rate is 1.0ml per minute;

The detection wavelength is 205nm;

The column temperature is 4°C;

The injection volume was 100 μl.

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

Further, in the described method, the need testing solution is: take the methionine sulfoxide reference substance, accurately weigh it, dissolve in water and quantitatively dilute to make a reference substance solution containing about 1mg of methionine sulfoxide in every 1ml, as methionine sulfoxide. Reference substance stock solution; take the methionine sulfone reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a reference substance solution containing about 1 mg of methionine sulfone per 1 ml, as the methionine sulfone reference substance stock solution; respectively accurately measure the compound amino acid for injection Liquid 18AA, methionine sulfoxide reference substance stock solution, methionine sulfone reference substance stock solution, 1ml each, mixed and placed in a 10ml volumetric flask, diluted with water to the mark, shaken up, and used as the test solution.

Further, in the method, a reference solution is prepared, and a high performance liquid chromatograph is used to detect the reference solution, taking the peak area as the ordinate and the injection concentration as the abscissa, performing linear regression, drawing a standard curve and obtaining a regression equation; The preparation of the control solution is as follows: accurately measure an appropriate amount of the methionine sulfoxide reference substance stock solution, dilute with water, and prepare solutions containing about 0.30, 5, 10, 50, 100 and 200 μg of methionine sulfoxide per 1 ml, respectively, as methionine sulfoxide. Sulfone reference solution: Precisely measure an appropriate amount of the methionine sulfone reference stock solution, dilute it with water, and prepare solutions containing about 4.20, 5, 10, 50, 100 and 200 μg of methionine sulfone per 1 ml, respectively, as the methionine sulfone control solution.

The present invention has the following advantages:

The detection method of the invention is simple and feasible, the analysis time is short, the specificity is strong, and the reproducibility is good, and the methionine sulfoxide and methionine sulfone impurities in the compound amino acid injection 18AA can be simultaneously separated and detected within 35 minutes, and the existing method is provided. The separation and determination problems that cannot be solved by the technology, so as to ensure the controllable quality of compound amino acid injection 18AA, and finally determine the safety and effectiveness of the product.

Description of drawings

Fig. 1 is the chromatogram of compound amino acid injection 18AA.

Figure 2 is the chromatogram of the test solution.

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

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

Figure 5 is a linear graph of methionine sulfoxide.

Figure 6 is a linear graph of methionine sulfone.

Figure 7 is the chromatogram of the test solution under normal conditions.

Figure 8 is the chromatogram of the test solution under the condition of flow rate + 10%.

Figure 9 is the chromatogram of the test solution under the condition of flow rate -10%.

Figure 10 is the chromatogram of the test solution under the condition of organic phase ratio + 1%.

Figure 11 is the chromatogram of the test solution under the condition of organic phase ratio-1%.

Figure 12 is the chromatogram of the test solution under the condition of mobile phase pH value + 0.05.

Figure 13 is the chromatogram of the test solution under the condition of mobile phase pH value -0.05.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below with reference to the preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

The reagents, medicines, instruments and equipment used in the specific embodiments of the present invention are all known products and can be obtained by purchasing commercially available products. details as follows:

Reagents and Medicines:

Methionine sulfoxide reference substance (arkpharminc, batch number: WG0002486-160825001); methionine sulfone reference substance (Beijing Bailingwei Technology Co., Ltd., batch number: LCC0Q84);

Compound amino acid injection (18AA) (Chenxin Pharmaceutical Co., Ltd., batch number: 1611052143);

Acetonitrile (TEDIA, batch number: AS1122-801) is chromatographically pure;

Diamine hydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd., batch number: 20170301)

Sodium octane sulfonate (Sahn Chemical Technology Shanghai Co., Ltd., batch number: EI050108) was of analytical grade.

equipment:

Waters1525 high performance liquid chromatograph;

Shanghai Lei magnetic desktop precision digital display PHS-3G acidity meter;

1/100,000 precision electronic balance model: AUW220D;

Circulating water vacuum pump model: SHB-Ⅲ.

Example 1

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

1) Prepare reference stock solution:

Take the methionine sulfoxide reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a reference substance solution containing about 1 mg of methionine sulfoxide per 1 ml, as the methionine sulfoxide reference substance stock solution.

Take the methionine sulfone reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a reference substance solution containing about 1 mg of methionine sulfone per 1 ml, as the methionine sulfone reference substance stock solution.

2) Prepare control solution

Precisely measure an appropriate amount of methionine sulfoxide reference substance stock solution, dilute with water, and prepare solutions containing about 0.30, 5, 10, 50, 100, and 200 μg of methionine sulfoxide per 1 ml, respectively, as methionine sulfoxide control solution (1), (2), (3), (4), (5) and (6).

Precisely measure an appropriate amount of methionine sulfone reference stock solution, dilute with water, and prepare solutions containing about 4.20, 5, 10, 50, 100, and 200 μg of methionine sulfone per 1 ml, respectively, as methionine sulfone control solutions (1), (2) , (3), (4), (5) and (6).

3) Prepare the test solution

Since the content of two impurities, methionine sulfoxide and methionine sulfone, in the compound amino acid injection 18AA sample is very small, it is not convenient to explore the chromatographic conditions. In order to facilitate the investigation of the chromatographic conditions, the test solution was prepared as follows:

Precisely measure 1ml each of Compound Amino Acid Injection 18AA, methionine sulfoxide reference substance stock solution, and methionine sulfone reference substance stock solution, mix them in a 10ml measuring bottle, add water to dilute to the mark, shake well, and use as the test solution.

4) Determination

Take methionine sulfoxide linear control solution (1)-(6), methionine sulfone linear control solution (1)-(6) and test solution, respectively, inject 100 μl into high performance liquid chromatograph, and record the chromatogram. Typical spectra are shown in Figures 1-4.

The chromatographic conditions are:

Chromatographic column: Titank C18, 4.6mm×250mm, 5μm;

Mobile phase A is 0.01mol·L ^-1 diammonium hydrogen phosphate solution, which contains 0.01mol·L ^-1 sodium octane sulfonate buffer solution, and the pH value is adjusted to 1.9±0.05 with phosphoric acid;

Mobile phase B: acetonitrile;

Carry out linear gradient elution according to Table 1, the flow rate is 1.0ml per minute;

Detection wavelength: 205nm;

Column temperature: 4℃;

Injection volume: 100 μl.

Table 1 Gradient elution procedure

time/min Mobile phase A/% Mobile phase B/% 0 100 0 twenty two 100 0 twenty three 90 10 29 90 10 30 100 0 35 100 0

In Figure 2, the retention time of 14.780 minutes is methionine sulfoxide, and the retention time of 16.465 minutes is methionine sulfone. The degree of separation between methionine sulfone and methionine sulfoxide is 1.8, and methionine sulfone and adjacent peaks and methionine sulfoxide and adjacent peaks are well separated. Figure 2 shows that the object of the present invention can be achieved under this condition.

Example 2 (Linear Range)

According to the preparation method of the methionine sulfoxide control solution and the methionine sulfone control solution in Example 1, the methionine sulfoxide control solutions (1), (2), (3), (4), (5) and (6) and Methionine sulfone control solutions (1), (2), (3), (4), (5) and (6).

Precisely measure methionine sulfoxide control solutions (1), (2), (3), (4), (5) and (6) and methionine sulfone control solutions (1), (2), (3), (4 ), (5) and (6), 100 μl each, respectively, were injected into the high performance liquid chromatograph, the chromatogram was recorded, and the peak area was measured. Take the peak area A as the ordinate and the injection concentration C as the abscissa, perform linear regression, and calculate the correlation coefficient (r).

Table 2 methionine sulfoxide linearity and range test results

Table 3 methionine sulfone linearity and range test results

The results showed that methionine sulfoxide was in the concentration range of 0.31-201.76 μg·ml ^-1 , and the linear curve was A=44217C-3774, r=1 (see Table 2 and Figure 5 for details). In the concentration range of methionine sulfone from 4.51 to 225.72 μg·ml ^-1 , the linear curve was A=2618C-1360, r=0.9999 (see Table 3 and Figure 6 for details). There was a significant linear relationship between the injection concentrations of methionine sulfoxide and methionine sulfone and their corresponding peak area response values.

Example 3 (precision)

1. Injection precision:

Precisely measure an appropriate amount of methionine sulfoxide reference substance stock solution, dilute it with water, and prepare a solution containing about 10 μg of methionine sulfoxide per 1 ml. The RSD was 0.16% (Table 4).

Precisely measure an appropriate amount of the methionine sulfone reference stock solution, dilute with water, and prepare a solution containing about 10 μg of methionine sulfone per 1 ml. Under the chromatographic conditions of Example 1, continuous injections were carried out 6 times, and the RSD of the peak area of methionine sulfone was 1.84. %(table 5). As can be seen from Tables 4 and 5, the repeatability was good.

Table 4 methionine sulfoxide linearity and range test results

Table 5 methionine sulfone linearity and range test results

injection sequence Peak area 1 28458 2 29655 3 29798 4 29329 5 29288 6 28637 average value 29341.4 RSD(%) 1.84

2. Repeatability test:

According to the method in Example 1, 6 parts of the test solution were prepared in parallel. Precisely measure 6 parts of the test solution, methionine sulfoxide control solution (1), (2), (3), (4), (5) and (6) and methionine sulfone control solution (1), (2) , (3), (4), (5) and (6), and analyzed by sample injection respectively, the average content of methionine sulfoxide was 1003.1 μg·ml ^-1 , the RSD was 0.13%; the average content of methionine sulfone was 1182.4 μg·ml ^-1 , the RSD was 1.41% (see Table 6 for details). The data results show that the method has good repeatability.

Table 6 Repeated experimental results

Example 4 (solution stability test)

The test solution was placed at room temperature, and measured after 0, 3, 6, 10, 14, 17, 20, and 24 hours, respectively. The injection volume was 100 μl. The main peak area and impurity content of the test solution were used to investigate the test. solution stability. The experimental results are shown in Table 7:

Table 7 Test results for the stability of the test solution

It can be seen from Table 7 that the test solution is stable within 24 hours at room temperature.

Example 5 (accuracy: sample recovery experiment)

Preparation of the solution:

Take the methionine sulfoxide (MetsoX) reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a reference substance solution containing about 1 mg of methionine sulfoxide per 1 ml, as the methionine sulfoxide reference substance stock solution.

Take the methionine sulfone (MetsoN) reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a reference substance solution containing about 1 mg of methionine sulfone per 1 ml, as the methionine sulfone reference substance stock solution.

According to the method in Example 1, respectively prepare methionine sulfoxide control solutions (1), (2), (3), (4), (5), (6) and methionine sulfone control solutions (1), (2), (3), (4), (5), (6).

Add 50μg·ml ^-1 , 100μg·ml ^-1 , 1000μg·ml ^-1 of methionine sulfoxide and methionine sulfone to compound amino acid injection (18AA) respectively (the specific preparation method is as follows: Precisely measure compound amino acid injection (18AA). ) 1ml, put it in a 10ml volumetric flask, add 1ml of methionine sulfoxide and methionine sulfone solutions with concentrations of about 50μg·ml ^-1 , 100μg·ml ^-1 , 1000μg·ml ^-1 respectively), each concentration point is parallel Three solutions were prepared, and the content of each of the above impurities was measured according to the measurement method of Example 1. Calculate the sample recovery and RSD of each impurity. The recovery results are shown in Table 8.

Table 8 Test results of sample addition recovery rate (n=9)

It can be seen from Table 8 that the recoveries of methionine sulfoxide (MetsoX) and methionine sulfone (MetsoN) in this method are respectively 99.09-102.14% and 98.20-101.21%, and the measurement accuracy is good.

Example 6 (durability test)

Appropriately adjust the flow rate of the mobile phase, the pH value of the mobile phase and the proportion of the organic phase, change the chromatographic conditions in Example 1 to a small extent, and compare the measurement results of methionine sulfoxide and methionine sulfone in the same sample under different conditions. The durability of the chromatographic conditions was investigated, the results are shown in Table 9, and the chromatograms are shown in Figures 7 to 13.

Figure 7 is the chromatogram of the test solution under normal conditions. Figure 8 is the chromatogram of the test solution under the condition of flow rate + 10%. Figure 9 is the chromatogram of the test solution under the condition of flow rate -10%. Figure 10 is the chromatogram of the test solution under the condition of organic phase ratio + 1%. Figure 11 is the chromatogram of the test solution under the condition of organic phase ratio-1%. Figure 12 is the chromatogram of the test solution under the condition of mobile phase pH value + 0.05. Figure 13 is the chromatogram of the test solution under the condition of mobile phase pH value -0.05.

Table 9 Durability test results

Note:- ^a : The resolution is not shown in the chromatogram.

It can be seen from Table 9 that after slight changes in the flow rate of the mobile phase, the pH value of the buffer salt, and the composition ratio of the mobile phase, under the condition of organic phase ratio + 1%, methionine sulfoxide (MetsoX) and methionine sulfone (MetsoN) were Adjacent peaks overlap without separation. Although the retention time of methionine sulfoxide (MetsoX) and methionine sulfone (MetsoN) changed slightly under the other conditions, the determination results were basically the same, and the determination durability of related substances was good. Therefore, it is necessary to strictly control the proportion of the organic phase to change within the specified range during the experiment.

The detection method of the present invention uses acidic ion pair reagent and organic solvent as mobile phases, and adopts high performance liquid chromatography to separate and measure methionine sulfoxide and methionine sulfone impurities in compound amino acid injection 18AA, and can simultaneously separate methionine sulfoxide within 35 minutes. The detection of sulfone and methionine sulfone impurities is very meaningful for the quality control of compound amino acid injection 18AA and the research on amino acid degradation pathway.

Obviously, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limiting the embodiments of the present invention. Changes or changes in other different forms cannot be exhausted here, and all obvious changes or changes derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims (5)

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 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.

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.

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