CN110441415B

CN110441415B - Arginine isomer detection method

Info

Publication number: CN110441415B
Application number: CN201910671653.5A
Authority: CN
Inventors: 刘超; 李斌水; 李晓微; 汪飞; 石江涛; 梁云科; 刘翠环; 李素霞; 李海松; 张亮; 武存景
Original assignee: Jing Jing Pharmaceutical Co ltd
Current assignee: Jing Jing Pharmaceutical Co ltd
Priority date: 2019-07-24
Filing date: 2019-07-24
Publication date: 2022-04-19
Anticipated expiration: 2039-07-24
Also published as: CN110441415A

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a detection method of arginine isomer. The detection method comprises the following steps: preparing a D-arginine reference substance into a reference substance aqueous solution, and preparing the reference substance aqueous solution and a derivative reagent into a reference substance solution; preparing an aqueous solution of a to-be-detected product from the to-be-detected product, and preparing the aqueous solution of the to-be-detected product and a derivative reagent into a solution of the to-be-detected product; and (3) respectively taking the reference substance solution and the solution of the to-be-detected substance, detecting by using a liquid chromatography, and calculating the content of the D-arginine in the to-be-detected substance by using an external standard method. The method for detecting the arginine isomer provided by the invention has the advantages of good specificity, strong sensitivity and high accuracy, and can effectively control the content of D-arginine in L-arginine and guarantee the quality of L-arginine products.

Description

Arginine isomer detection method

Technical Field

The invention relates to the technical field of biological medicines, in particular to a detection method of arginine isomer.

Background

L-arginine has L-type and D-type, L-arginine exists naturally, the production method of L-arginine is mainly a microbial fermentation method, and the production of D-arginine in the production process is difficult to avoid. In order to improve and ensure the quality of the L-arginine product, the content of the D-arginine must be strictly controlled.

At present, a detection method for content of isomers in arginine is rarely reported, and arginine is alpha-amino acid and is absorbed by a tail end under ultraviolet wavelength, so that the content of D-arginine cannot be sensitively and accurately controlled in the production process of L-arginine.

Disclosure of Invention

Aiming at the problems that the content of D-arginine is difficult to be sensitively and accurately measured in the existing L-arginine production process, so that the quality of an L-arginine product is influenced and the like, the invention provides a method for detecting an arginine isomer.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

a method for detecting arginine isomer, comprising the following steps:

(1) preparing a D-arginine reference substance into a reference substance aqueous solution, and preparing the reference substance aqueous solution and a derivative reagent into a reference substance solution;

(2) preparing an aqueous solution of a to-be-detected product from the to-be-detected product, and preparing the aqueous solution of the to-be-detected product and a derivative reagent into a solution of the to-be-detected product;

(3) respectively taking the reference substance solution and the solution of the to-be-detected substance, detecting by liquid chromatography, and calculating the content of D-arginine in the to-be-detected substance by adopting an external standard method;

the chromatographic conditions of the liquid chromatogram are as follows: the wavelength is 225-235 nm; a chromatographic column: octadecylsilane chemically bonded silica chromatographic column; the column temperature is 25-35 ℃; mobile phase: the mobile phase A is 0.05mol/L ammonium acetate solution-acetonitrile, and the mobile phase B is acetonitrile; the sample injection temperature is 2-8 ℃, and linear gradient elution is carried out, wherein the linear elution gradient is as follows: 0 min: 100% A + 0% B; 20 min: 100% A + 0% B; 35 min: 80% A + 20% B; and (5) 37 min: 80% A + 20% B; 37.1 min: 100% A + 0% B; and (4) 45 min: 100% A + 0% B.

Compared with the prior art, the method for detecting the arginine isomer, provided by the invention, is characterized in that an aqueous solution of a to-be-detected product containing both L-arginine and D-arginine is mixed with a derivatization reagent to prepare a solution of the to-be-detected product, so that the arginine and the derivatization reagent are subjected to derivatization reaction, and a group with strong ultraviolet absorption is introduced into an arginine molecule, so that the detection wavelength is changed into 225-235nm (corresponding to strong ultraviolet absorption), thereby overcoming the difficulty that the liquid phase condition cannot be effectively detected due to the terminal absorption of arginine, and improving the detection sensitivity; meanwhile, the derivative method is adopted, so that the derivatives of the L-arginine and the D-arginine can be effectively separated, the separation degree of the arginine isomer under the chromatographic condition is improved by detecting the arginine derivative, and the detection accuracy is improved; in addition, under the condition of 2-8 ℃, linear gradient elution is adopted, so that the stability of peak area is ensured, the separation degree of arginine isomers is further improved, and the detection accuracy is improved. The method can effectively control the content of D-arginine in L-arginine and ensure the quality of L-arginine products.

Further, the derivatization reagent is prepared by mixing a mixture of o-phthalaldehyde and N-acetyl-L-cysteine with a boric acid buffer solution in a molar ratio of 1-1.05: 1. The o-phthalaldehyde and the N-acetyl-L-cysteine are subjected to derivatization reaction with arginine in a molar ratio of 1:1, so that the reaction is thorough, impurity interference caused by excessive derivatization reagents is avoided, the o-phthalaldehyde is slightly excessive, and the sensitivity and accuracy of detection are improved.

Further, the molar ratio of the D-arginine to the N-acetyl-L-cysteine in the reference solution is 0.001: 1-1.5; the molar ratio of arginine to N-acetyl-L-cysteine in the solution of the sample to be detected is 1: 1-1.5. The appropriate excess of derivatizing reagent is selected to ensure efficient derivatization and to avoid significant blank (derivatizing reagent) interference.

Further, the detection wavelength of the gradient detection is 230 nm. The arginine derivative has maximum absorption at the position, has stronger ultraviolet absorption by taking 230nm as the detection wavelength, can avoid the tail end absorption of arginine and can improve the detection sensitivity.

Further, the volume ratio of the 0.05mol/L ammonium acetate solution to the acetonitrile in the mobile phase A is 85-95: 5-15. Due to the fact that the ammonium acetate solution is 0.05mol/L, when the pH value of the measured solution is close to 6, the chromatographic column is prone to bacterium growing and damage is caused, the mobile phase A is adjusted to be 0.05mol/L ammonium acetate solution-acetonitrile, damage to the chromatographic column and equipment caused by the mobile phase can be effectively avoided, normal running of detection is facilitated, and cost is reduced.

Furthermore, the sample injection volume is 10-20 μ l and the flow rate is 0.5-2ml/min in the detection process. Ensures effective separation of arginine isomer in the linear gradient elution process, and is beneficial to improving the detection accuracy of arginine isomer.

Further, the sample injection temperature in the detection process is 5 ℃. The peak area of the control solution at 5 ℃ was changed as shown in Table 1, and the peak stability at 5 ℃ was excellent and could be stably maintained for 15 hours.

TABLE 1

Further, the boric acid buffer solution is a boric acid-sodium hydroxide buffer solution, and the pH value is 8-10. The boric acid-sodium hydroxide buffer solution is prepared by 0.2mol/L boric acid solution and sodium hydroxide. The method is used for adjusting the pH value of the system, preventing the dissociation of the sample, facilitating the separation of arginine isomer and improving the detection accuracy.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

A method for detecting arginine isomer, comprising the following steps:

(1) accurately weighing 80mg of o-phthalaldehyde and 93mg of N-acetyl-L-cysteine, placing the o-phthalaldehyde and the N-acetyl-L-cysteine into the same 25ml volumetric flask, adding 2.5ml of ethanol for dissolving, adding a boric acid buffer solution with the pH value of 9.8 for diluting to a scale, and performing constant volume to obtain a derivative reagent;

(2) precisely weighing a proper amount of a D-arginine reference substance, and preparing a reference substance water solution with the concentration of 1 mu g/ml. Precisely measuring 2ml of the reference substance aqueous solution and 0.6ml of the derivative reagent, placing the reference substance aqueous solution and the derivative reagent in the same 5ml volumetric flask, adding a boric acid buffer solution to dilute to a scale, shaking up, and fixing the volume to obtain a reference substance solution;

(3) accurately weighing 10mg of the to-be-detected product, placing the to-be-detected product in a 10ml volumetric flask, adding water to dissolve and dilute the to-be-detected product to a scale, shaking up, fixing the volume and filtering to obtain the to-be-detected product aqueous solution. Precisely measuring 2ml of the aqueous solution of the to-be-measured product and 0.6ml of the derivative reagent, putting the aqueous solution of the to-be-measured product and the derivative reagent into the same 5ml volumetric flask, adding a boric acid buffer solution to dilute the aqueous solution of the to-be-measured product to a scale, shaking up the solution, and fixing the volume to obtain the solution of the to-be-measured product;

(4) and (4) taking the reference substance solution and the solution to be detected respectively, and performing gradient detection by using liquid chromatography. Chromatographic conditions for gradient detection were: a chromatographic column: octadecylsilane chemically bonded silica chromatographic column; the column temperature is 30 ℃; mobile phase: the mobile phase A is 0.05mol/L ammonium acetate solution-acetonitrile (90:10), and the mobile phase B is acetonitrile; the sample injection temperature is 5 ℃, the sample injection volume is 15 μ l, the flow rate is lml/min, and the linear gradient elution is shown in table 2:

TABLE 2

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
20	100	0
			35	80	20
37	80	20
			37.1	100	0
45	100	0

Recording chromatogram data, and calculating the content (Cx) of D-arginine in the sample to be tested by adopting an external standard method.

Wherein, Cr is the content of a reference substance; ar is the peak area of the reference; ax is the peak area of D-arginine in the sample to be detected.

Example 2

A method for detecting arginine isomer, comprising the following steps:

(1) accurately weighing 80mg of o-phthalaldehyde and 93mg of N-acetyl-L-cysteine, placing the o-phthalaldehyde and the N-acetyl-L-cysteine into the same 25ml volumetric flask, adding 2.5ml of ethanol for dissolving, adding a boric acid buffer solution with the pH value of 8 for diluting to a scale, and performing constant volume to obtain a derivative reagent;

(2) precisely weighing a proper amount of a D-arginine reference substance, and preparing a reference substance water solution with the concentration of 1 mu g/ml. Precisely measuring 2ml of the reference substance aqueous solution and 0.5ml of the derivative reagent, placing the reference substance aqueous solution and the derivative reagent in the same 5ml volumetric flask, adding a boric acid buffer solution to dilute to a scale, shaking up, and fixing the volume to obtain a reference substance solution;

(3) accurately weighing 10mg of the to-be-detected product, placing the to-be-detected product in a 10ml volumetric flask, adding water to dissolve and dilute the to-be-detected product to a scale, shaking up, fixing the volume and filtering to obtain the to-be-detected product aqueous solution. Precisely measuring 2ml of the aqueous solution of the to-be-measured product and 0.5ml of the derivative reagent, putting the aqueous solution of the to-be-measured product and the derivative reagent into the same 5ml volumetric flask, adding a boric acid buffer solution to dilute the aqueous solution of the to-be-measured product to a scale, shaking up the solution, and fixing the volume to obtain the solution of the to-be-measured product;

(4) and (4) taking the reference substance solution and the solution to be detected respectively, and performing gradient detection by using liquid chromatography. Chromatographic conditions for gradient detection were: a chromatographic column: octadecylsilane chemically bonded silica chromatographic column; the column temperature was 25 ℃; mobile phase: the mobile phase A is 0.05mol/L ammonium acetate solution-acetonitrile (95:5), and the mobile phase B is acetonitrile; the sample injection temperature was 2 ℃, the sample injection volume was 10 μ l, and the flow rate was 0.5ml/min, and a linear gradient elution was performed, the linear elution gradient being shown in table 3:

TABLE 3

Example 3

A method for detecting arginine isomer, comprising the following steps:

(1) accurately weighing 80mg of o-phthalaldehyde and 93mg of N-acetyl-L-cysteine, placing the o-phthalaldehyde and the N-acetyl-L-cysteine into the same 25ml volumetric flask, adding 2.5ml of ethanol for dissolving, adding a boric acid buffer solution with the pH value of 10 for diluting to a scale, and performing constant volume to obtain a derivative reagent;

(2) precisely weighing a proper amount of a D-arginine reference substance, and preparing a reference substance water solution with the concentration of 1 mu g/ml. Precisely measuring 2ml of the reference substance aqueous solution and 0.75ml of the derivative reagent, placing the reference substance aqueous solution and the derivative reagent in the same 5ml volumetric flask, adding a boric acid buffer solution to dilute to a scale, shaking up, and fixing the volume to obtain a reference substance solution;

(3) accurately weighing 10mg of the to-be-detected product, placing the to-be-detected product in a 10ml volumetric flask, adding water to dissolve and dilute the to-be-detected product to a scale, shaking up, fixing the volume and filtering to obtain the to-be-detected product aqueous solution. Precisely measuring 2ml of the aqueous solution of the to-be-measured product and 0.75ml of the derivative reagent, putting the aqueous solution of the to-be-measured product and the derivative reagent into the same 5ml volumetric flask, adding a boric acid buffer solution to dilute the aqueous solution of the to-be-measured product to a scale, shaking up the solution, and fixing the volume to obtain the solution of the to-be-measured product;

(4) and (4) taking the reference substance solution and the solution to be detected respectively, and performing gradient detection by using liquid chromatography. Chromatographic conditions for gradient detection were: a chromatographic column: octadecylsilane chemically bonded silica chromatographic column; the column temperature was 35 ℃; mobile phase: the mobile phase A is 0.05mol/L ammonium acetate solution-acetonitrile (85:15), and the mobile phase B is acetonitrile; the sample injection temperature was 8 ℃, the sample injection volume was 20 μ l, and the flow rate was 2ml/min, and a linear gradient elution was performed, the linear elution gradient being shown in table 4:

TABLE 4

To better illustrate the characteristics of the method for detecting arginine isomers provided in the embodiment of the present invention, the following method in embodiment 1 is used to test 9 sets of samples to be detected (process batches C011707048, C01170707049, C01170707050, C0117070707070707051, C0117070707052, C011707053, C011707096, C011707097, and C011707098), and the results are shown in table 5.

TABLE 5

Batch number	Isomer (D-arginine) content
		C011707048	0.026
C011707049	0.021
		C011707050	0.014
C011707051	0.039
		C011707052	0.057
C011707053	0.045
		C011707096	0.018
C011707097	0.029
		C011707098	0.025

The data show that the content of isomers in 9 groups of samples to be detected is less than 0.1%, the batch-to-batch difference is small, and the detection method provided by the embodiment of the invention can sensitively and accurately detect the isomers in arginine and effectively control the quality of the L-arginine product. The detection methods in embodiments 2 and 3 of the present invention can achieve effects equivalent to those of the detection method in embodiment 1.

In order to better illustrate the characteristics of the detection method of arginine isomer provided by the embodiment of the present invention, the specificity, sensitivity, accuracy and linear relationship between the content of D-arginine and the peak area of the detection method provided by the embodiment of the present invention are verified below.

The specificity of the detection method is as follows:

accurately weighing 80mg of o-phthalaldehyde and 93mg of N-acetyl-L-cysteine, placing the o-phthalaldehyde and the N-acetyl-L-cysteine into the same 25ml volumetric flask, adding 2.5ml of ethanol for dissolving, adding a boric acid buffer solution with the pH value of 9.8 for diluting to a scale, and performing constant volume to obtain the derivative reagent.

Taking a proper amount of a D-arginine reference substance and a proper amount of L-arginine, adding water to dissolve and dilute the D-arginine reference substance and the L-arginine reference substance to prepare a mixed solution containing about 1 mu g of D-arginine and 1mg of L-arginine in each lml, precisely measuring 2ml of the mixed solution and 0.6ml of a derivative reagent, putting the mixed solution and the derivative reagent into the same 5ml volumetric flask, adding a boric acid buffer solution with the pH value of 9.8 to dilute to a scale, shaking up and fixing the volume to obtain a system applicability solution.

2ml of precision water and 0.6ml of derivatization reagent are put into the same 5ml volumetric flask, and then boric acid buffer solution with pH of 9.8 is added to dilute the solution to the scale, and the solution is shaken up to be used as blank solution.

The chromatographic conditions used in example 1 were used for the detection. The results are shown in Table 6.

TABLE 6

The result shows that the blank solution has no interference to the determination of the D-arginine, and the D-arginine derivative peak and the L-arginine derivative peak can be effectively separated, which shows that the detection method has better specificity.

Sensitivity of the detection method:

preparing a D-arginine standard solution, and diluting to prepare a series of solutions with a series of concentrations. The detection limit is determined according to the signal-to-noise ratio, and a control solution with a known concentration is diluted to a low concentration, and the concentration of S/N-3 is used as a detection limit solution. The detection limit of the D-arginine is 0.021 mg/L. The detection method provided by the embodiment of the invention has higher sensitivity.

Accuracy of the detection method:

accurately weighing 80mg of o-phthalaldehyde and 93mg of N-acetyl-L-cysteine, placing the o-phthalaldehyde and the N-acetyl-L-cysteine into the same 25ml volumetric flask, adding 2.5ml of ethanol for dissolving, adding a boric acid buffer solution (the same below) with the pH value of 9.8 for diluting to a scale, and performing constant volume to obtain the derivative reagent.

Precisely weighing a proper amount of a D-arginine reference substance, and preparing a reference substance water solution with the concentration of 1 mu g/ml. Precisely measuring 2ml of the reference substance aqueous solution and 0.6ml of the derivative reagent, placing the reference substance aqueous solution and the derivative reagent in the same 5ml volumetric flask, adding a boric acid buffer solution to dilute to a scale, shaking up, and fixing the volume to obtain the reference substance solution.

Precisely weighing 10mg of D-arginine reference substance, placing the D-arginine reference substance in a 10ml volumetric flask, adding water to dissolve and dilute the D-arginine reference substance to scale, shaking up, precisely weighing 1ml, placing the D-arginine reference substance in a 100ml volumetric flask, adding water to dilute the D-arginine reference substance to scale, and shaking up to obtain D-arginine reference substance storage solution.

Accurately weighing 10mg of the to-be-detected product, placing the to-be-detected product in a 10ml volumetric flask, adding water to dissolve and dilute the to-be-detected product to a scale, shaking up, fixing the volume and filtering to obtain the to-be-detected product aqueous solution. Precisely measuring 2ml of the aqueous solution of the to-be-measured product and 0.6ml of the derivative reagent, putting the aqueous solution of the to-be-measured product and the derivative reagent into the same 5ml volumetric flask, adding a boric acid buffer solution to dilute the aqueous solution of the to-be-measured product to a scale, shaking up the solution, and fixing the volume to obtain the solution of the to-be-measured product.

And (3) precisely weighing three parts of a to-be-detected product, each part of which is 10mg, respectively, placing the three parts into three 10ml volumetric flasks, precisely weighing 0.8ml of the D-arginine reference product stock solution respectively, placing the stock solution into the volumetric flasks, adding water to dissolve the stock solution, diluting the stock solution to a scale, and shaking up. And respectively and precisely measuring 2ml of the solution and 0.6ml of the derivatization reagent, putting the solution and the derivatization reagent into the same 5ml measuring flask, adding a boric acid buffer solution to dilute the solution to a scale, and shaking up the solution to obtain a solution with the recovery rate of 80%.

And (3) precisely weighing three parts of a to-be-detected product, each part of which is 10mg, respectively, placing the three parts into three 10ml volumetric flasks, precisely weighing 1ml of the D-arginine reference product stock solution respectively, placing the stock solution into the volumetric flasks, adding water to dissolve the stock solution, diluting the stock solution to a scale, and shaking up the stock solution. And respectively and precisely measuring 2ml of the solution and 0.6ml of the derivatization reagent, putting the solution and the derivatization reagent into the same 5ml measuring flask, adding a boric acid buffer solution to dilute the solution to a scale, and shaking up the solution to obtain the solution with the recovery rate of 100%.

And precisely weighing three parts of a to-be-detected product, each part of which is 10mg, respectively, placing the three parts into three 10ml volumetric flasks, precisely weighing 1.2ml of the D-arginine reference product stock solution respectively, placing the stock solution into the volumetric flasks, adding water to dissolve the stock solution, diluting the stock solution to a scale, and shaking up the stock solution. And precisely measuring 2ml of the solution and 0.6ml of the derivatization reagent respectively, placing the solution and the derivatization reagent in the same 5ml measuring flask, adding a boric acid buffer solution to dilute the solution to a scale, and shaking up the solution to obtain a solution with the recovery rate of 120%.

And precisely measuring the reference solution, the solution to be detected and the recovery rate solution by 15 mu l respectively, injecting into a liquid chromatograph for detection, and recording a chromatogram. The content of D-arginine was calculated by peak area according to the external standard method. The results are shown in Table 7.

TABLE 7

From the data, the three horizontal recovery rate ranges are 94.8% -101.4%, and are all between 90% -108%, and the RSD of 9 recovery rate sample data is 2.99%, and is less than 3%, which indicates that the detection method provided by the invention has good accuracy.

The linear relation between the D-arginine content and the peak area is as follows:

precisely measuring the D-arginine reference substance stock solutions 0.5ml, 0.8ml, 1ml, 1.5ml and 2.0ml respectively, placing in different 10ml volumetric flasks, adding water to dilute to scale, shaking up and fixing the volume. Precisely measuring the constant volume solutions respectively 2ml, placing in different 5ml volumetric flasks, respectively adding 0.6ml of derivatization reagent, diluting to scale with boric acid buffer solution with pH of 9.8, and shaking up. As linear 50%, 80%, 100%, 150%, 200% solutions.

The results of linear regression using the D-arginine concentration as the abscissa (x) and the peak area as the ordinate (y) are shown in Table 8.

TABLE 8

As can be seen from the data in the table, the linear relation of the D-arginine is good in the range of 0.062 mu g/ml to 0.821 mu g/ml, and the detection accuracy and the reliability are ensured. The linear equation is that Y is 171.33x +1.1695, the correlation coefficient r is 0.9998, the Y-axis intercept is 1.1695, the response value is 1.6 percent of 100 percent, the RSD of the response factor is 3.94 percent and is less than 10 percent, and the requirement is met.

The data show that the method for detecting the arginine isomer provided by the invention has the advantages of good specificity, strong sensitivity and high accuracy, and can effectively control the content of D-arginine in L-arginine and ensure the product quality of the L-arginine.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The method for detecting the arginine isomer is characterized by comprising the following steps:

the chromatographic conditions of the liquid chromatogram are as follows: wavelength 225-; a chromatographic column: octadecylsilane chemically bonded silica chromatographic column; the column temperature is 25-35 ℃; mobile phase: the mobile phase A is 0.05mol/L ammonium acetate solution-acetonitrile, and the mobile phase B is acetonitrile; the sample injection temperature is 2-8 ℃, and linear gradient elution is carried out, wherein the linear elution gradient is as follows: 0 min: 100% A + 0% B; 20 min: 100% A + 0% B; 35 min: 80% A + 20% B; and (5) 37 min: 80% A + 20% B; 37.1 min: 100% A + 0% B; and (4) 45 min: 100% A + 0% B;

the volume ratio of 0.05mol/L ammonium acetate solution to acetonitrile in the mobile phase A is 85-95: 5-15;

the derivatization reagent is prepared from a mixture of o-phthalaldehyde and N-acetyl-L-cysteine with a molar ratio of 1-1.05:1 and a boric acid buffer solution.

2. The method for detecting an arginine isomer according to claim 1, which comprises: the molar ratio of the D-arginine to the N-acetyl-L-cysteine in the reference solution is 0.001: 1-1.5; the molar ratio of arginine to N-acetyl-L-cysteine in the solution of the sample to be detected is 1: 1-1.5.

3. The method for detecting an arginine isomer according to claim 1, which comprises: the detection wavelength of the gradient detection is 230 nm.

4. The method for detecting an arginine isomer according to claim 1, which comprises: the sample injection volume is 10-20 μ l and the flow rate is 0.5-2ml/min in the detection process.

5. The method for detecting an arginine isomer according to claim 1, which comprises: the boric acid buffer solution is boric acid-sodium hydroxide buffer solution, and the pH value is 8-10.