阅读说明：本技术 一种乙酰半胱氨酸对映异构体的分离纯化方法 (Separation and purification method of acetylcysteine enantiomer ) 是由 李敬 黄蕾 杨磊 梅雪臣 何韺 刘珍 吕凡林 于 2019-09-20 设计创作，主要内容包括：本发明公开了一种乙酰半胱氨酸对映异构体的分离纯化方法。该分离纯化方法包括下述步骤：采用高效液相色谱法对N-乙酰半胱氨酸对映异构体进行分离,即可；所述的高效液相色谱法中的流动相为烷烃类溶剂-异丙醇,所述烷烃类溶剂和所述异丙醇的体积比为(70～83):(17～30)；所述的高效液相色谱法中的色谱柱的填料为涂覆有如式I所示的纤维素类衍生物的填料。本发明所提供的分离纯化方法可以将N-乙酰-L-半胱氨酸及其对映异构体进行有效分离,进一步实现了对N-乙酰-L-半胱氨酸及其对映异构体的定性、定量分析,对药品生产和质量提升有重要指导意义。<Image he="333" wi="629" file="DDA0002209194840000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention discloses a method for separating and purifying acetylcysteine enantiomer. The separation and purification method comprises the following steps: separating N-acetylcysteine enantiomer by high performance liquid chromatography; the mobile phase in the high performance liquid chromatography is alkane solvent-isopropanol, and the volume ratio of the alkane solvent to the isopropanol is (70-83) to (17-30); the filler of the chromatographic column in the high performance liquid chromatography is coated with cellulose derivatives shown in a formula I. The separation and purification method provided by the invention can separate N-acetylThe effective separation of the L-cysteine and the enantiomer thereof further realizes the qualitative and quantitative analysis of the N-acetyl-L-cysteine and the enantiomer thereof, and has important guiding significance for the production and quality improvement of medicines.)

1. A method for separating and purifying N-acetylcysteine enantiomer is characterized by comprising the following steps: separating N-acetylcysteine enantiomer by high performance liquid chromatography;

the mobile phase in the high performance liquid chromatography is alkane solvent-isopropanol, and the volume ratio of the alkane solvent to the isopropanol is (70-83) to (17-30);

the filler of the chromatographic column in the high performance liquid chromatography is coated with cellulose derivatives shown in formula I;

wherein R is

2. The method for separating and purifying N-acetylcysteine enantiomer of claim 1 wherein R is₁Is H;

and/or, said R₂Is C₁～C₃Preferably methyl or ethyl;

and/or, said R₃Is H;

and/or, said R₄Is C₁～C₃Preferably methyl or ethyl;

and/or, said R₅Is H; preferably, R is

And/or the matrix of the filler is spherical silica gel;

and/or the particle size of the filler is 4-6 μm, preferably 5 μm.

3. The method for separating and purifying N-acetylcysteine enantiomer of claim 1 wherein the alkane solvent is one or more of N-hexane, N-pentane, dichloromethane and chloroform, preferably N-hexane.

4. The method for separating and purifying N-acetylcysteine enantiomer of claim 1 wherein the isopropanol contains acid and/or tetrahydrofuran.

5. The method for separating and purifying N-acetylcysteine enantiomer according to claim 4, wherein when the isopropanol contains an acid, the acid is acetic acid;

and/or, when the isopropanol contains acid, the amount of the acid is 0.1-0.5%, preferably 0.1% or 0.5%, and the percentage refers to the volume percentage in the isopropanol;

and/or, when the isopropanol contains tetrahydrofuran, the amount of the tetrahydrofuran is 0.05-0.3%, preferably 0.05-0.15%, more preferably 0.1%, and the percentage refers to the volume percentage in the isopropanol.

6. The method for separating and purifying N-acetylcysteine enantiomer of claim 5, wherein the mobile phase is alkane solvent-isopropanol containing 0.1-0.5% acid or alkane solvent-isopropanol containing 0.05-0.3% tetrahydrofuran, preferably alkane solvent-isopropanol containing 0.1% acid, alkane solvent-isopropanol containing 0.5% acid or alkane solvent-isopropanol containing 0.1% tetrahydrofuran;

or, the alkane solvent-isopropanol containing 0.1-0.5% of acid and 0.05-0.3% of tetrahydrofuran, preferably the alkane solvent-isopropanol containing 0.5% of acid and 0.1% of tetrahydrofuran; percentages refer to volume percent in the isopropyl alcohol.

7. The method for separating and purifying N-acetylcysteine enantiomer of claim 1, wherein the volume ratio of the alkane solvent to the isopropanol is (75-83) - (17-25), preferably 77:23, 80:20 or 83: 17.

8. The method for separating and purifying N-acetylcysteine enantiomer according to any one of claims 1-7, wherein the flow rate of the mobile phase is 0.8-1.2 ml/min, preferably 1.0 ml/min;

and/or in the high performance liquid chromatography, the sample injection amount is 15-25 mu L, preferably 20 mu L;

and/or the length of the chromatographic column is 100-250 mm, preferably 250 mm;

and/or the temperature of the chromatographic column is 25-40 ℃, preferably 25 ℃ or 30 ℃;

and/or the detector of the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 220 nm.

9. Use of the method of any one of claims 1 to 8 for the separation and purification of N-acetylcysteine enantiomer in the determination of the content of N-acetyl-L-cysteine and/or N-acetyl-D-cysteine, preferably, the solution to be tested is diluted and then subjected to the content determination, wherein the dilution is chromatographic grade absolute ethanol.

10. Use according to claim 9, wherein the content determination is carried out by a standard curve method; preferably, the standard curve method comprises the following steps:

(1) taking an N-acetyl-L-cysteine reference substance solution and/or an N-acetyl-D-cysteine reference substance solution, and carrying out isovolumetric sample injection under the same chromatographic condition with the solution to be detected; measuring peak areas of the reference solutions at different concentrations to obtain a concentration-peak area standard curve;

(2) and calculating the concentration of the N-acetyl-L-cysteine and/or the concentration of the N-acetyl-D-cysteine in the solution to be detected according to the measured peak area of the solution to be detected.

Technical Field

The invention relates to the field of drug analysis and detection, and particularly relates to a separation and purification method of acetylcysteine enantiomer.

Background

N-acetyl-L-cysteine (Ac-Cys), molecular formula: c₅H₉NO₃S, molecular weight: 163.19. the product is white crystalline powder; odor similar to garlic and sour taste; it has hygroscopicity. Easily soluble in water, soluble in ethanol, insoluble in dichloromethane and diethyl ether; the structural formula is shown as follows.

N-acetyl-L-cysteine is a mucolytic agent, is suitable for dyspnea caused by a large amount of viscous phlegm obstruction and expectoration difficulty diseases, can also be used for patients with phlegm viscosity and expectoration difficulty caused by postoperative expectoration difficulty, acute and chronic bronchitis, bronchiectasis, pneumonia, pulmonary tuberculosis, emphysema and the like, and is also mainly used for treating idiopathic interstitial lung. In recent years, it has been found that a 3% aqueous solution of the medicine can be used as an eye drop (acimetion) for treating keratitis, and with the research and development of fine cosmetics, recent photomedical and photobiological studies show that N-acetyl-L-cysteine can reduce subcutaneous melanin and eliminate the deposited melanin, and has been increasingly paid attention as a whitening cosmetic.

N-acetyl-L-cysteine is widely applied to the fields of medicines, pesticides, chemical industry and the like, and currently, only the conventional physicochemical index of N-acetyl-L-cysteine is detected, but a byproduct, namely N-acetyl-D-cysteine, which is an enantiomer is generated in the production process, so that the product quality is influenced.

HPLC generally has three methods for resolving amino acid racemates: firstly, a chiral reagent and a resolved substance are subjected to derivatization reaction to generate diastereoisomers, so that the diastereoisomers can be resolved by a traditional achiral chromatographic column; secondly, adding a chiral additive into the mobile phase, and utilizing an achiral stationary phase chromatographic column to perform resolution; thirdly, the chiral stationary phase chromatographic column is utilized for resolution. Wherein the derivatization reaction is complex in operation, and the derivatization reagent and the reaction medium are likely to interfere with chromatographic separation and detection; the chiral mobile phase additive method has a complex resolution mechanism and is difficult to establish, and the system can be balanced for a long time when the mobile phase is replaced.

At present, the absolute content of enantiomers of drugs with chiral characteristics is increasingly represented by ee values, chiral analysis of N-acetylcysteine enantiomers generally adopts a derivatization method for separation, the operation is complex, and the reaction degree in the derivatization process is difficult to accurately control, so that the content of N-acetyl-L-cysteine and N-acetyl-D-cysteine is difficult to accurately determine, and the reproducibility is poor.

Therefore, a separation and purification method capable of quickly and accurately measuring the content of acetylcysteine enantiomer is established, and the method has important guiding significance for medicine production and quality improvement.

Disclosure of Invention

The invention aims to solve the technical problems of complex separation and purification method of N-acetylcysteine enantiomer, inaccurate quantification caused by difficult accurate control of derivatization reaction degree and poor reproducibility in the prior art, and provides a separation and purification method of acetylcysteine enantiomer. The separation and purification method provided by the invention can effectively separate the N-acetyl-L-cysteine and the enantiomer thereof, and further realizes qualitative and quantitative analysis of the N-acetyl-L-cysteine and the enantiomer thereof. Based on the method, the method can simply, quickly and accurately analyze the enantiomer impurity N-acetyl-D-cysteine generated in the synthesis process of the N-acetyl-L-cysteine, and has important guiding significance for the production and quality improvement of medicines.

In the research and development process, the inventor finds that N-acetyl-L-cysteine and enantiomers thereof can be effectively separated by using cellulose to replace a derivatized coating type chiral chromatographic column and using a normal phase mixed solvent as a flowing phase to perform separation detection on the enantiomers of acetylcysteine.

The invention provides a method for separating and purifying an N-acetylcysteine enantiomer, which comprises the following steps: separating N-acetylcysteine enantiomer by high performance liquid chromatography;

the mobile phase in the high performance liquid chromatography is alkane solvent-isopropanol, and the volume ratio of the alkane solvent to the isopropanol is (70-83) to (17-30);

the filler of the chromatographic column in the high performance liquid chromatography is coated with cellulose derivatives shown in formula I;

wherein R is

R₁、R₂、R₃、R₄And R₅Independently is H or C₁～C₃Alkyl group of (1).

In the present invention, R is₁Preferably H.

In the present invention, R is₂Preferably C₁～C₃More preferably methyl or ethyl, such as methyl.

In the present invention, R is₃Preferably H.

In the present invention, R is₄Preferably C₁～C₃More preferably methyl or ethyl, such as methyl.

In the present invention, R is₅Preferably H.

In the present invention, R is preferably

In the invention, the cellulose derivative shown as the formula I is preferably cellulose-tri (3,5) dimethyl phenyl carbamate.

In the present invention, n is the degree of polymerization of cellulose and is generally a positive integer.

In the present invention, the matrix of the filler may be a filler matrix conventional in the art, such as spherical silica gel.

In the present invention, the particle size of the filler may be a particle size conventional in the art, for example, 4 to 6 μm, and further for example, 5 μm.

In the present invention, the column may be referred to as a coating-type chiral column, depending on the nature of the packing.

In the present invention, the alkane solvent may be an alkane solvent that is conventional in the art and can be used as a normal phase solvent in chromatographic separation, for example, one or more of n-hexane, n-pentane, dichloromethane and chloroform, preferably n-hexane.

In the present invention, preferably, the isopropyl alcohol contains an acid and/or tetrahydrofuran.

Wherein the acid may be acetic acid.

Wherein the acid may be used in an amount conventional in the art, such as 0.1 to 0.5%, and further such as 0.1% or 0.5%, percentage referring to volume percentage in the isopropanol.

The amount of tetrahydrofuran is preferably 0.05-0.3%, such as 0.05-0.15%, and more preferably 0.1%, wherein the percentage refers to the volume percentage in the isopropanol.

In the present invention, the mobile phase is preferably an alkane solvent-isopropanol containing 0.1-0.5% of acid or an alkane solvent-isopropanol containing 0.05-0.3% of tetrahydrofuran, such as an alkane solvent-isopropanol containing 0.1% of acid, an alkane solvent-isopropanol containing 0.5% of acid or an alkane solvent-isopropanol containing 0.1% of tetrahydrofuran; more preferably an alkane solvent-isopropanol containing 0.1-0.5% acid, 0.05-0.3% tetrahydrofuran, such as an alkane solvent-isopropanol containing 0.5% acid, 0.1% tetrahydrofuran; percentages refer to volume percent in the isopropyl alcohol.

When the isopropanol contains tetrahydrofuran, the spontaneous formation of acetylcysteine self-cyclized compound (2 methyl-2 thiazoline-4 carboxylic acid, detailed in European pharmacopoeia 7.0) can be inhibited, and the influence of the cyclized compound on the quantification is reduced.

In the present invention, the mobile phase is preferably n-hexane-isopropanol, more preferably n-hexane-isopropanol containing 0.1 to 0.5% of acid, n-hexane-isopropanol containing 0.05 to 0.3% of tetrahydrofuran or n-hexane-isopropanol containing 0.1 to 0.5% of acid, 0.05 to 0.3% of tetrahydrofuran, for example n-hexane-isopropanol containing 0.1% of acetic acid, n-hexane-isopropanol containing 0.5% of acetic acid, n-hexane-isopropanol containing 0.1% of tetrahydrofuran or n-hexane-isopropanol containing 0.5% of acid, 0.1% of tetrahydrofuran, the percentages being volume percentages in the isopropanol.

In the present invention, preferably, the mobile phase is a mixed solution of the alkane solvent and the isopropyl alcohol.

When the mobile phase is a mixed solution of the alkane solvent and the isopropanol, the acid and/or tetrahydrofuran contained in the isopropanol can also be added into the alkane solvent after conversion according to percentage and then mixed with the isopropanol.

In the present invention, the volume ratio of the alkane solvent to the isopropanol is preferably (75-83): (17-25), for example 77:23, 80:20 or 83: 17.

In the present invention, the flow rate of the mobile phase is preferably 0.8 to 1.2ml/min, for example, 1.0 ml/min.

In the invention, the sample volume of the high performance liquid chromatography can refer to the conventional sample volume of HPLC detection and analysis in the field, such as 15-25 μ L, and further such as 20 μ L.

In the invention, the length of the chromatographic column can be 100-250 mm, such as 250 mm.

In the present invention, the temperature of the chromatography column is preferably 25 to 40 ℃, for example 25 ℃ or 30 ℃.

In the present invention, preferably, the detector of the high performance liquid chromatography may be an ultraviolet detector. The detection wavelength of the ultraviolet detector may be 220 nm.

In the present invention, the high performance liquid chromatograph in the high performance liquid chromatography may adopt a high performance liquid chromatograph conventional in the art, such as hitachi L2420 liquid chromatograph.

The invention also provides application of the separation and purification method of the N-acetylcysteine enantiomer in the content measurement of N-acetyl-L-cysteine and N-acetyl-D-cysteine.

In the content measurement, the solution to be measured can be diluted. The diluent may be chromatographic grade absolute ethanol.

The method for measuring the content of N-acetyl-L-cysteine and N-acetyl-D-cysteine can refer to content measuring methods conventional in the field, such as a standard curve method.

Preferably, the standard curve method comprises the following steps:

(1) taking an N-acetyl-L-cysteine reference substance solution and/or an N-acetyl-D-cysteine reference substance solution, and carrying out isovolumetric sample injection under the same chromatographic condition with the solution to be detected; measuring peak areas of the reference solutions at different concentrations to obtain a concentration-peak area standard curve;

(2) and calculating the concentration of the N-acetyl-L-cysteine and/or the N-acetyl-D-cysteine in the solution to be detected according to the measured peak area of the solution to be detected. On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

(1) the separation and purification method provided by the invention is simple to operate, can effectively separate the N-acetyl-L-cysteine and the enantiomer thereof, and further realizes qualitative and quantitative analysis of the N-acetyl-L-cysteine and the enantiomer thereof.

(2) The separation and purification method provided by the invention can be used for separating the N-acetyl-DL-cysteine, and can also be used for simply, quickly and accurately separating impurities generated in the synthesis process of the N-acetyl-L-cysteine. In addition, the separation and purification method of the invention effectively inhibits N-acetyl-L-cysteine from generating self-cyclized compound 2 methyl-2 thiazoline-4 carboxylic acid by adding tetrahydrofuran in the mobile phase, and the percent of the cyclized compound is less than or equal to 0.33 percent.

Drawings

FIG. 1 is a chromatogram of the solvent absolute ethanol in example 2.

FIG. 2 is a chromatogram of the 1 st injection of the mixed solution of N-acetyl-DL-cysteine standard in example 2, in which the peak 1 is N-acetyl-L-cysteine and the peak 2 is N-acetyl-D-cysteine.

FIG. 3 is a chromatogram of the 2 nd sample injection of the mixed solution of N-acetyl-DL-cysteine standard in example 2, in which peak 1 is N-acetyl-L-cysteine, peak 2 is N-acetyl-D-cysteine, and peak 3 is an impurity peak.

FIG. 4 is a chromatogram of the 3 rd sample injection of the mixed solution of N-acetyl-DL-cysteine standard in example 2, in which peak 1 is N-acetyl-L-cysteine, peak 2 is N-acetyl-D-cysteine, and peak 3 is an impurity peak.

FIG. 5 is a chromatogram of the 4 th sample injection of the mixed solution of N-acetyl-DL-cysteine standard substance in example 2, in which peak 1 is N-acetyl-L-cysteine, peak 2 is N-acetyl-D-cysteine, and peak 3 is an impurity peak.

FIG. 6 is a chromatogram of the 5 th sample injection of the mixed solution of N-acetyl-DL-cysteine standard substance in example 2, in which peak 1 is N-acetyl-L-cysteine, peak 2 is N-acetyl-D-cysteine, and peak 3 is an impurity peak.

FIG. 7 is a chromatogram of racemized N-acetyl-DL-cysteine in example 3, in which peak 1 is N-acetyl-L-cysteine, peak 2 is N-acetyl-D-cysteine, and peak 3 is an impurity peak.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.