阅读说明：本技术 异氰酸酯衍生物的分离检测方法 (Separation detection method of isocyanate derivative ) 是由 彭斯维 聂翠翠 高曼 乔春莲 于 2020-05-25 设计创作，主要内容包括：本发明提出了甲苄基异氰酸酯体的检测方法,所述方法包括：将所述含有甲苄基异氰酸酯的溶液进行高效液相色谱检测,以便于检测甲苄基异氰酸酯中的S-(-)-α-甲苄基异氰酸酯与R-(+)-α-甲苄基异氰酸酯。本发明的检测方法可以有效地分离出S-(-)-α-甲苄基异氰酸酯与R-(+)-α-甲苄基异氰酸酯,从而确定溶液中是否存在S-(-)-α-甲苄基异氰酸酯和/或R-(+)-α-甲苄基异氰酸酯及各自含量。该方法具有准确性强、灵敏度高、稳定性强、操作简便、快捷、效率高等优点,适于规模化应用,尤其适用于筛选可检测盐酸多佐胺异构体的甲苄基异氰酸酯试剂,从而实现对盐酸多佐胺异构体的检测。(The invention provides a detection method of methylbenzyl isocyanate, which comprises the following steps: and (3) carrying out high performance liquid chromatography detection on the solution containing the methylbenzyl isocyanate so as to detect S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate in the methylbenzyl isocyanate. The detection method can effectively separate S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate so as to determine whether S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate exist in the solution and the content of the S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate. The method has the advantages of high accuracy, high sensitivity, high stability, simplicity and convenience in operation, rapidness, high efficiency and the like, is suitable for large-scale application, and is particularly suitable for screening the methyl benzyl isocyanate reagent capable of detecting the dorzolamide hydrochloride isomer, so that the dorzolamide hydrochloride isomer can be detected.)

1. A method for detecting methylbenzyl isocyanate is characterized by comprising the following steps:

and carrying out high performance liquid chromatography detection on the solution containing the methylbenzyl isocyanate so as to be convenient for separating and detecting S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate in the methylbenzyl isocyanate, wherein a chromatographic column adopted by the high performance liquid chromatography detection is a bonding type chiral column.

2. The method of claim 1, wherein the high performance liquid chromatography detection comprises:

and performing gradient elution on the solution containing the methylbenzyl isocyanate by using a mobile phase, wherein the mobile phase A is selected from a mixed solution containing n-hexane, ethanol and diethylamine, the mobile phase B is selected from ethanol, and preferably, the volume ratio of the n-hexane, the ethanol and the diethylamine in the mobile phase A is (90-99): (1-10): (0.05-0.2).

3. The method of claim 2, wherein the conditions of the gradient elution are as follows:

4. the method according to claim 1, wherein the HPLC assay uses a column selected from the group consisting of YMC Cellulose-SB bonded chiral column and xylonite CHRALPAK IB chiral column, preferably the HPLC assay uses a YMC Cellulose-SB bonded chiral column, more preferably the column has a size of 4.6 x 250mm, 5 μm.

5. The method according to claim 1, wherein the detection wavelength used in the high performance liquid chromatography detection is 250-260 nm, and the column temperature is 10-20 ℃.

6. The process according to claim 1, characterized in that the process for obtaining the solution containing methylbenzyl isocyanate comprises:

diluting chemicals containing S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate by using a diluting solvent,

the dilution solvent is selected from a mixed solvent containing n-hexane and isopropanol, and preferably, the volume ratio of the n-hexane to the isopropanol in the dilution solvent is (85-95): (5-15).

7. The method of claim 1, further comprising:

and (2) detecting the content of S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate in the solution containing methylbenzyl isocyanate by adopting an external standard method.

8. A method for screening a methylbenzyl isocyanate reagent, which is characterized by comprising the following steps:

detecting a candidate methylbenzyl isocyanate reagent by using the methylbenzyl isocyanate detection method of any one of claims 1 to 7, and determining the content of R- (+) -alpha-methylbenzyl isocyanate and/or S- (-) -alpha-methylbenzyl isocyanate in the candidate methylbenzyl isocyanate reagent;

when the content of the R- (+) -alpha-methylbenzyl isocyanate is lower than a first threshold value and/or the content of the S- (-) -alpha-methylbenzyl isocyanate is higher than a second threshold value, the candidate methylbenzyl isocyanate reagent is an indication that the candidate methylbenzyl isocyanate reagent is a target reagent;

preferably, the first threshold is not greater than 1 mass%, more preferably not greater than 0.1 mass%;

the second threshold value is not less than 99 mass%, more preferably not less than 99.9 mass%.

9. The use of the method for detecting methylbenzyl isocyanate according to any one of claims 1 to 7 or the method for screening methylbenzyl isocyanate reagents according to claim 8 in the detection of dorzolamide hydrochloride isomers.

10. A method for detecting dorzolamide hydrochloride enantiomers, which is characterized by comprising the following steps:

1) screening a satisfactory reagent of methylbenzyl isocyanate by the method of claim 8;

2) carrying out derivatization reaction on the dorzolamide hydrochloride sample by adopting the methylbenzyl isocyanate reagent screened in the step 1) to obtain a derivatization product;

3) and detecting the derivative product, and determining whether the dorzolamide hydrochloride enantiomer exists in the dorzolamide hydrochloride sample and/or the dorzolamide hydrochloride enantiomer content based on the detection result.

Technical Field

The present invention relates to the field of analytical testing in chemistry. In particular, the invention relates to a separation detection method of isocyanate derivatives.

Background

The structural formula of the S- (-) -alpha-methylbenzyl isocyanate is shown as follows:

s- (-) -alpha-methylbenzyl isocyanate is generally used as a derivatizing agent or a resolving agent, and when the S- (-) -alpha-methylbenzyl isocyanate is used as a derivatizing agent, the existence or the content of isomers is very important for the detection accuracy. For example, S- (-) - α -methylbenzyl isocyanate can react with an enantiomer of dorzolamide hydrochloride (referred to as impurity a for short) to form a derivative of impurity a, and the derivative is separated and detected by high performance liquid chromatography, so that it can be used as a derivatization reagent for detecting dorzolamide hydrochloride isomer impurity a in pharmacopoeia of various countries. However, if the S- (-) - α -methylbenzyl isocyanate contains the optical isomer R- (+) - α -methylbenzyl isocyanate, then the derivative of R- (+) - α -methylbenzyl isocyanate and dorzolamide hydrochloride that forms the aforementioned impurity a is also detected by chromatographic separation, which undoubtedly interferes with the detection of the content of the impurity a in dorzolamide hydrochloride, resulting in the measured content of the impurity a in dorzolamide hydrochloride not matching the actual content. Therefore, in order to accurately detect the content of the impurity a in dorzolamide hydrochloride, it is necessary to separate S- (-) - α -methylbenzyl isocyanate from R- (+) - α -methylbenzyl isocyanate, and further determine whether R- (+) - α -methylbenzyl isocyanate exists in S- (-) - α -methylbenzyl isocyanate or select S- (-) - α -methylbenzyl isocyanate having a relatively low isomer content.

S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate are optical isomers, and a certain technical difficulty exists in separating the S- (-) -alpha-methylbenzyl isocyanate and the R- (+) -alpha-methylbenzyl isocyanate. In addition, the prior art does not disclose a method capable of effectively separating and detecting S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate.

Therefore, it is necessary to develop a method for detecting S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate with high separation degree and good stability.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a detection method of methylbenzyl isocyanate, a method for screening a methylbenzyl isocyanate reagent, application of the methylbenzyl isocyanate reagent in dorzolamide hydrochloride isomer detection and a detection method of dorzolamide hydrochloride isomers. The method has the advantages of high accuracy, high sensitivity, high stability, simplicity and convenience in operation, rapidness, high efficiency and the like, is suitable for large-scale application, and is particularly suitable for screening the methyl benzyl isocyanate reagent capable of detecting the dorzolamide hydrochloride isomer, so that the dorzolamide hydrochloride isomer can be detected.

In one aspect of the invention, the invention provides a method for detecting methylbenzyl isocyanate. According to an embodiment of the invention, the method comprises: and (3) carrying out high performance liquid chromatography detection on the solution containing the methylbenzyl isocyanate so as to be convenient for separating and detecting S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate in the methylbenzyl isocyanate, wherein a chromatographic column adopted by the high performance liquid chromatography detection is a bonding type chiral column.

According to the detection method provided by the embodiment of the invention, S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate can be effectively separated by adopting high performance liquid chromatography detection, and the separation degree is up to more than 1.0, even more than 1.5, so that whether S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate exist in the methylbenzyl isocyanate solution can be determined. The method has the advantages of high accuracy, high sensitivity, high stability, simplicity and convenience in operation, rapidness, high efficiency and the like, and is suitable for large-scale application.

According to the embodiment of the present invention, the detection method of methylbenzyl isocyanate may further have the following additional technical features:

according to an embodiment of the invention, the high performance liquid chromatography detection comprises: and performing gradient elution on the solution containing the methylbenzyl isocyanate by using a mobile phase, wherein the mobile phase A is selected from a mixed solution containing n-hexane, ethanol and diethylamine, the mobile phase B is selected from ethanol, and preferably, the volume ratio of the n-hexane, the ethanol and the diethylamine in the mobile phase A is (90-99): (1-10): (0.05-0.2).

According to an embodiment of the invention, the conditions of the gradient elution are as follows:

according to an embodiment of the present invention, the hplc assay uses a column selected from a YMC Cellulose-SB bonded chiral column (product brand is YMC/vimesin, product model is Cellulose-SB) or a gorskil chraplak IB chiral column (product brand is cellosolve, product model is chraplak IB), and preferably, the hplc assay uses a column selected from a YMC Cellulose-SB bonded chiral column with a size of 4.6 × 250mm, 5 μm.

According to the embodiment of the invention, the detection wavelength adopted in the high performance liquid chromatography detection is 250-260 nm, and the column temperature is 10-20 ℃.

According to an embodiment of the present invention, the method for obtaining said solution containing methylbenzyl isocyanate comprises: diluting a chemical containing S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate by using a diluting solvent, wherein the diluting solvent is selected from a mixed solvent containing n-hexane and isopropanol, and preferably, in the diluting solvent, the volume ratio of n-hexane to isopropanol is (85-95): (5-15).

According to an embodiment of the invention, the method further comprises: and (2) detecting the content of S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate in the solution containing methylbenzyl isocyanate by adopting an external standard method.

In yet another aspect of the present invention, the present invention provides a method for screening methylbenzyl isocyanate reagents. According to an embodiment of the invention, the method comprises: detecting the candidate methylbenzyl isocyanate reagent by adopting the detection method of the methylbenzyl isocyanate, and determining the content of R- (+) -alpha-methylbenzyl isocyanate and/or S- (-) -alpha-methylbenzyl isocyanate in the candidate methylbenzyl isocyanate reagent; when the content of the R- (+) -alpha-methylbenzyl isocyanate is lower than a first threshold value and/or the content of the S- (-) -alpha-methylbenzyl isocyanate is higher than a second threshold value, the candidate methylbenzyl isocyanate reagent is an indication that the candidate methylbenzyl isocyanate reagent is a target reagent. Therefore, the method can accurately screen out the methylbenzyl isocyanate reagent with the content of R- (+) -alpha-and/or S- (-) -alpha-methylbenzyl isocyanate meeting the requirement, avoids interference caused by excessive R- (+) -alpha-methoxy isocyanate contained in the reagent, and particularly can be used for screening out the methylbenzyl isocyanate reagent capable of detecting dorzolamide hydrochloride isomers.

According to an embodiment of the present invention, the first threshold is not more than 1 mass%, more preferably not more than 0.1 mass%; the second threshold value is not less than 99 mass%, more preferably not less than 99.9 mass%.

In another aspect of the invention, the invention provides the application of the detection method of the methyl benzyl isocyanate or the method for screening the methyl benzyl isocyanate reagent in the detection of dorzolamide hydrochloride isomers. As mentioned above, the detection method of methylbenzyl isocyanate can effectively separate R- (+) -alpha-and S- (-) -alpha-methylbenzyl isocyanate and further determine the respective contents, so that a methylbenzyl isocyanate reagent meeting the content requirement of R- (+) -alpha-and/or S- (-) -alpha-methylbenzyl isocyanate is screened out to be applied to the detection of dorzolamide hydrochloride isomers.

In another aspect of the invention, the invention provides a method for detecting dorzolamide hydrochloride isomers. According to an embodiment of the invention, the method comprises: 1) screening out the methyl benzyl isocyanate reagent meeting the requirement by utilizing the method for screening the methyl benzyl isocyanate reagent; 2) carrying out derivatization reaction on the dorzolamide hydrochloride sample by adopting the methylbenzyl isocyanate reagent screened in the step 1) to obtain a derivatization product; 3) and detecting the derivative product, and determining whether the dorzolamide hydrochloride isomer exists in the dorzolamide hydrochloride sample and/or determining the dorzolamide hydrochloride isomer content based on the detection result. Therefore, the method provided by the embodiment of the invention can effectively realize the detection of dorzolamide hydrochloride isomers, has the advantages of strong accuracy, high sensitivity, strong stability, simplicity and convenience in operation, quickness, high efficiency and the like, and is suitable for large-scale application.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a high performance liquid chromatogram of a test solution in example 1 according to the present invention;

FIG. 2 shows a high performance liquid chromatogram of a solution I (for localization) of a R- (+) -alpha-methylbenzyl isocyanate standard in example 1 according to the present invention;

FIG. 3 shows a high performance liquid chromatogram of a S- (-) - α -methylbenzyl isocyanate standard solution in example 1 according to the present invention;

FIG. 4 shows a high performance liquid chromatogram of an empty white solution according to example 1 of the present invention;

FIG. 5 shows a high performance liquid chromatogram of a test solution in example 2 according to the present invention;

FIG. 6 shows a high performance liquid chromatogram of a test solution in example 3 according to the present invention;

FIG. 7 shows a high performance liquid chromatogram of the test solution in comparative example 1 according to the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

The detection method of the methylbenzyl isocyanate, the method for screening the methylbenzyl isocyanate reagent, the application of the reagent in the detection of the dorzolamide hydrochloride isomer, and the detection method of the dorzolamide hydrochloride isomer are respectively described in detail below.

Detection method of methylbenzyl isocyanate

In one aspect of the invention, the invention provides a method for detecting methylbenzyl isocyanate. According to an embodiment of the invention, the method comprises: and (2) carrying out high performance liquid chromatography detection on the solution containing the methylbenzyl isocyanate so as to be convenient for separating and detecting S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate in the methylbenzyl isocyanate, wherein a chromatographic column adopted by the high performance liquid chromatography detection is a bonding type chiral column.

According to the detection method provided by the embodiment of the invention, S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate can be effectively separated by adopting high performance liquid chromatography detection, and the separation degree is up to more than 1.0, even more than 1.5, so that whether S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate exist in the methylbenzyl isocyanate solution can be determined. The method has the advantages of high accuracy, high sensitivity, high stability, simplicity and convenience in operation, rapidness, high efficiency and the like, and is suitable for large-scale application.

According to an embodiment of the present invention, the high performance liquid chromatography detection comprises: and (3) carrying out gradient elution on the solution containing the methylbenzyl isocyanate by using a mobile phase, wherein the mobile phase A is selected from a mixed solution containing n-hexane, ethanol and diethylamine, and the mobile phase B is selected from ethanol.

The inventor adopts high performance liquid chromatography to detect S- (-) -alpha-and R- (+) -alpha-methylbenzyl isocyanate, and finds that the composition of a mobile phase can significantly influence whether the S- (-) -alpha-and R- (+) -alpha-methylbenzyl isocyanate can be separated obviously, and if the mobile phase is not selected properly, the S- (-) -alpha-and R- (+) -alpha-methylbenzyl isocyanate cannot be separated obviously, so that whether the solution contains the S- (-) -alpha-and R- (+) -alpha-methylbenzyl isocyanate cannot be judged accurately, and the specific content is further difficult to determine. Furthermore, the inventor discovers through in-depth research analysis and optimization screening that S- (-) -alpha-and R- (+) -alpha-methylbenzyl isocyanate can be effectively separated and detected by adopting n-hexane, ethanol and diethylamine as a mobile phase A and ethanol as a mobile phase B, the separation degree is high, the problems that the peak area cannot be calculated due to poor peak shape, the separation degree is low or the peaks are overlapped to cause the effective separation and the like can be avoided, the detection result is high in accuracy, high in sensitivity and high in stability, and the method is simple, convenient and rapid to operate, high in efficiency and suitable for large-scale application.

According to the embodiment of the invention, the volume ratio of n-hexane, ethanol and diethylamine in the mobile phase A is (90-99): (1-10): (0.05-0.2). The inventor obtains the better proportion through a large number of experiments, so that S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate can be further effectively separated, the problems that peak areas cannot be calculated due to poor peak shapes, the separation degree is low or the peaks are overlapped to cause the problem that the peaks cannot be effectively separated and the like are solved, and the accuracy, the sensitivity and the stability of detection results are improved.

According to an embodiment of the present invention, the conditions of gradient elution are as follows. Therefore, the S- (-) -alpha-methylbenzyl isocyanate and the R- (+) -alpha-methylbenzyl isocyanate can be further effectively separated, and the accuracy, the sensitivity and the stability of a detection result are improved.

According to the embodiment of the invention, the high performance liquid chromatography detection adopts a chromatographic column selected from YMC Cellulose-SB bonded chiral columns or xylonite CHRALPAK IB chiral columns. The two chromatographic columns can be used for further effectively separating S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate, so that the problems that peak areas cannot be calculated due to poor peak shapes, the separation degree is low or the peaks are overlapped to cause the problem that the peaks cannot be effectively separated and the like are solved, and the accuracy, the sensitivity and the stability of detection results are improved. Wherein, the YMC Cellulose-SB bonding type chiral column (with the size of 4.6 × 250mm and the size of 5 μm) has better effect.

According to the embodiment of the invention, the detection wavelength adopted in the chromatographic detection is 250-260 nm, and the column temperature is 10-20 ℃. Therefore, S- (-) -alpha-and R- (+) -alpha-methylbenzyl isocyanate can be further and effectively separated, and the accuracy, the sensitivity and the stability of a detection result are improved.

According to an embodiment of the present invention, a method of obtaining a solution containing methylbenzyl isocyanate comprises: diluting a chemical containing S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate by using a diluting solvent, wherein the diluting solvent is selected from a mixed solvent containing n-hexane and isopropanol.

The inventor finds that the composition of a diluting solvent influences the separation of S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate, and further, the inventor finds that the S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate can be effectively separated during chromatographic detection by taking a mixed solvent containing n-hexane and isopropanol as the diluting solvent, so that the problems that the peak area cannot be calculated due to poor peak shape, the separation degree is low or the peaks overlap together to cause the ineffective separation and the like are avoided, and the accuracy, the sensitivity and the stability of the detection result are improved.

According to the embodiment of the invention, in the diluting solvent, the volume ratio of n-hexane to isopropanol is (85-95): (5-15). Therefore, the S- (-) -alpha-methylbenzyl isocyanate and/or the R- (+) -alpha-methylbenzyl isocyanate can be further and effectively separated, the problems that peak areas cannot be calculated due to poor peak shapes, the separation degree is low or the peaks are overlapped to cause the problem that the peaks cannot be effectively separated and the like are solved, and the accuracy, the sensitivity and the stability of detection results are improved.

According to an embodiment of the invention, the method further comprises: and (3) detecting the content of S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate in the solution containing methylbenzyl isocyanate by adopting an external standard method. Therefore, the content of the S- (-) -alpha-methylbenzyl isocyanate and/or the R- (+) -alpha-methylbenzyl isocyanate can be accurately determined, and the aim of quantitative detection is fulfilled.

Method for screening methylbenzyl isocyanate reagent

In yet another aspect of the present invention, the present invention provides a method for screening methylbenzyl isocyanate reagents. According to an embodiment of the invention, the method comprises: detecting the candidate methylbenzyl isocyanate reagent by adopting the detection method of the methylbenzyl isocyanate, and determining the content of R- (+) -alpha-methylbenzyl isocyanate and/or S- (-) -alpha-methylbenzyl isocyanate in the candidate methylbenzyl isocyanate reagent; when the content of R- (+) -alpha-methylbenzyl isocyanate is lower than a first threshold value and/or the content of S- (-) -alpha-methylbenzyl isocyanate is higher than a second threshold value, the candidate methylbenzyl isocyanate reagent is an indication that the candidate methylbenzyl isocyanate reagent is a target reagent. Therefore, the method can accurately screen out the methylbenzyl isocyanate reagent with the content of R- (+) -alpha-and/or S- (-) -alpha-methylbenzyl isocyanate meeting the requirement, avoids the interference caused by excessive R- (+) -alpha-methoxy isocyanate contained in the reagent, and particularly can be used for screening out the methylbenzyl isocyanate reagent capable of detecting the enantiomer (impurity A for short) of dorzolamide hydrochloride.

It should be noted that the methylbenzyl isocyanate described in the present invention is to be understood in a broad sense, and may be R- (+) - α -methylbenzyl isocyanate, or a mixture of R- (+) - α -methylbenzyl isocyanate and S- (-) - α -methylbenzyl isocyanate in any ratio. The methylbenzyl isocyanate reagent described in the invention is also to be understood in a broad sense, and all the reagents containing methylbenzyl isocyanate can be regarded as methylbenzyl isocyanate reagents, the content of R- (+) -alpha-methylbenzyl isocyanate and S- (-) -alpha-methylbenzyl isocyanate in the methylbenzyl isocyanate reagent is not strictly required, and the methylbenzyl isocyanate reagent with a specific composition can be selected according to actual needs. In addition, the specific values of the first threshold and the second threshold are not strictly limited, and can be flexibly selected according to actual needs, for example, the first threshold may be not more than 1 vol%, and preferably not more than 0.1 vol%; the second threshold value is not less than 99% by volume, preferably not less than 99.9% by volume. Therefore, the content of S- (-) -alpha-methoxy isocyanate in the screened methylbenzyl isocyanate reagent is high, and the method is particularly suitable for detecting the enantiomer (impurity A for short) of dorzolamide hydrochloride.

It will be appreciated by those skilled in the art that the features and advantages described above for the method of detecting methylbenzyl isocyanate are equally applicable to the method of screening for methylbenzyl isocyanate reagents and will not be described in detail herein.

Application of detection method of methylbenzyl isocyanate or method for screening methylbenzyl isocyanate reagent in dorzolamide hydrochloride isomer detection

In another aspect of the invention, the invention provides the application of the detection method of the methyl benzyl isocyanate or the method for screening the methyl benzyl isocyanate reagent in the detection of dorzolamide hydrochloride isomers. As mentioned above, the detection method for screening methylbenzyl isocyanate can effectively separate R- (+) -alpha-and S- (-) -alpha-methylbenzyl isocyanate and further determine the contents of the R- (+) -alpha-and S- (-) -alpha-methylbenzyl isocyanate, so that the methylbenzyl isocyanate reagent meeting the content requirement of the R- (+) -alpha-and/or S- (-) -alpha-methylbenzyl isocyanate is screened out and applied to the detection of dorzolamide hydrochloride isomers. Specifically, S- (-) - α -methoxyisocyanate can be reacted with an enantiomer of dorzolamide hydrochloride (referred to as impurity a for short) to produce a derivatizing agent of impurity a, which can be further detected by, for example, high performance liquid chromatography. The method can effectively screen out the methylbenzyl isocyanate reagent which hardly contains R- (+) -alpha-methylbenzyl isocyanate, so that the adverse effect of the increase of the detection content of the impurity A caused by the derivatization reaction of the R- (+) -alpha-methylbenzyl isocyanate and the impurity A can be greatly reduced.

It will be appreciated by those skilled in the art that the features and advantages described above for the method of detecting methylbenzyl isocyanate and the method of screening for methylbenzyl isocyanate reagents apply equally to this application and will not be described in any further detail herein.

Method for detecting dorzolamide hydrochloride isomer

In another aspect of the invention, the invention provides a method for detecting dorzolamide hydrochloride isomers. According to an embodiment of the invention, the method comprises: 1) screening out the methyl benzyl isocyanate reagent meeting the requirement by utilizing the method for screening the methyl benzyl isocyanate reagent; 2) carrying out derivatization reaction on the dorzolamide hydrochloride sample by adopting the methylbenzyl isocyanate reagent screened in the step 1) to obtain a derivatization product; 3) and detecting the derivative product, and determining whether the dorzolamide hydrochloride enantiomer exists in the dorzolamide hydrochloride sample and/or the dorzolamide hydrochloride enantiomer content based on the detection result.

As mentioned above, the method for screening the methylbenzyl isocyanate reagent can effectively screen the methylbenzyl isocyanate reagent which hardly contains R- (+) -alpha-methylbenzyl isocyanate, so that the adverse effect of the increase of the detection content of the impurity A caused by the derivatization reaction of the R- (+) -alpha-methylbenzyl isocyanate and the impurity A can be greatly reduced. Therefore, the method provided by the embodiment of the invention can effectively realize the detection of dorzolamide hydrochloride isomers, has the advantages of strong accuracy, high sensitivity, strong stability, simplicity and convenience in operation, quickness, high efficiency and the like, and is suitable for large-scale application.

It will be appreciated by those skilled in the art that the features and advantages described above for the method of screening methylbenzyl isocyanate reagents are equally applicable to the method of detecting dorzolamide hydrochloride isomers and will not be described in detail herein.

The invention has the beneficial effects that:

according to the methylbenzyl isocyanate detection method provided by the embodiment of the invention, S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate can be effectively separated by adopting high performance liquid chromatography detection, and the separation degree is up to more than 1.0, even more than 1.5, so that whether S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate exist in the methylbenzyl isocyanate-containing solution can be determined. And the content of S- (-) -alpha-methylbenzyl isocyanate and/or R- (+) -alpha-methylbenzyl isocyanate can be further determined by using a mode such as an external standard method, so that the aim of quantitative detection is fulfilled. The method has the advantages of high accuracy, high sensitivity, high stability, simplicity and convenience in operation, rapidness, high efficiency and the like, is suitable for large-scale application, and particularly can be used for screening out a methylbenzyl isocyanate reagent meeting requirements, such as a methylbenzyl isocyanate reagent with low R- (+) -alpha-methylbenzyl isocyanate content, and the reagent can be used for realizing the detection of dorzolamide hydrochloride enantiomers.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

Chromatographic conditions are as follows:

a chromatographic column: YMC Cellulose-SB (4.6 mm, 5 μm);

a detector: a UV detector;

detection wavelength: 254 nm;

column temperature: 10 ℃;

diluting the solvent: n-hexane-isopropanol in a volume ratio of 90: 10;

mobile phase A: n-hexane-ethanol-diethylamine with a volume ratio of 90:10: 0.05;

mobile phase B: ethanol;

gradient elution conditions:

the measurement method is as follows:

blank solution: i.e. the diluent solvent.

Preparing a test solution: 200 mu g of S- (-) -alpha-methylbenzyl isocyanate test sample reagent (sold in the market) is weighed, placed in a 10mL measuring flask, diluted to the scale by adding a diluting solvent, and shaken up to obtain the test sample.

S- (-) -alpha-methylbenzyl isocyanate standard solution: measuring 200 mu g of S- (-) -alpha-methylbenzyl isocyanate standard substance, placing the standard substance into a 10mL measuring flask, adding a diluting solvent to dilute to a scale, and shaking up to obtain the product.

A first R- (+) -alpha-methylbenzyl isocyanate standard solution (for positioning): weighing 200 mu g of R- (+) -alpha-methylbenzyl isocyanate standard substance, placing the standard substance into a 10mL measuring flask, adding a diluting solvent to dilute to a scale, and shaking up to obtain the product.

R- (+) -alpha-methylbenzyl isocyanate standard stock solution: weighing 10mg of R- (+) -alpha-methylbenzyl isocyanate standard substance, placing the standard substance into a 100mL measuring flask, adding a diluting solvent to dilute to a scale, and shaking up to obtain the product.

A R- (+) -alpha-methylbenzyl isocyanate standard substance solution II (for quantification): weighing 50 mu L of R- (+) -alpha-methylbenzyl isocyanate stock solution, placing the R- (+) -alpha-methylbenzyl isocyanate stock solution into a 20mL measuring flask, adding a diluting solvent to dilute to a scale, and shaking up to obtain the product.

Precisely measuring 10 mu L of a blank solution, a test sample solution, an S- (-) -alpha-methylbenzyl isocyanate standard substance solution, a R- (+) -alpha-methylbenzyl isocyanate standard substance solution I and a R- (+) -alpha-methylbenzyl isocyanate standard substance solution II, respectively injecting into a high performance liquid chromatograph, and recording a chromatogram, wherein the flow rate is 0.6ml/min, and the chromatogram is shown in figures 1 to 4.

As can be seen from FIG. 1, the peak-off time of S- (-) - α -methylbenzyl isocyanate was about 17.55 minutes, and the peak-off time of R- (+) - α -methylbenzyl isocyanate was about 16.67 minutes. The degrees of separation of S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate were 1.58.

The content (%) of R- (+) - α -methylbenzyl isocyanate was (a-like × C-pair)/(a-pair × C-like) × 100%

In the above formula:

the peak area of R- (+) - α -methylbenzyl isocyanate in the sample a-sample solution was specifically 1.584 in this example;

the peak area of R- (+) - α -methylbenzyl isocyanate in the standard solution of p-R- (+) - α -methylbenzyl isocyanate (ii) in this example is specifically 1.059;

the concentration (μ g/mL) of R- (+) - α -methylbenzyl isocyanate in the standard solution (ii) of C-p-R- (+) - α -methylbenzyl isocyanate, specifically 0.25 μ g/mL in this example;

the concentration of the sample C-sample solution (. mu.g/mL), specifically 20. mu.g/mL in this example.

The content of the impurity R- (+) -alpha-methylbenzyl isocyanate in the test solution is calculated according to the formula as follows: 1.87 percent. The R- (+) -alpha-methylbenzyl isocyanate can be accurately determined by adopting the high performance liquid chromatography conditions.

Example 2

Chromatographic conditions are as follows:

a chromatographic column: YMC Cellulose-SB (4.6 mm, 5 μm);

a detector: a UV detector;

detection wavelength: 260 nm;

column temperature: 20 ℃;

diluting the solvent: n-hexane-isopropanol in a volume ratio of 85: 15;

mobile phase A: n-hexane-ethanol-diethylamine with a volume ratio of 95:5: 0.1;

mobile phase B: ethanol;

gradient elution conditions: as in example 1.

Preparing a test solution, a S- (-) -alpha-methylbenzyl isocyanate standard solution, a R- (+) -alpha-methylbenzyl isocyanate standard solution I, a R- (+) -alpha-methylbenzyl isocyanate standard solution II and a blank solution according to the determination method described in example 1, and recording chromatograms, wherein FIG. 5 is a high performance liquid chromatogram of the test solution.

As can be seen from FIG. 5, the peak-off time of S- (-) - α -methylbenzyl isocyanate was about 17.37 minutes, and the peak-off time of R- (+) - α -methylbenzyl isocyanate was about 16.55 minutes. The degrees of separation of S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate were 1.24.

Example 3

Chromatographic conditions are as follows:

a chromatographic column: YMC Cellulose-SB (4.6 mm, 5 μm);

a detector: a UV detector;

detection wavelength: 250 nm;

column temperature: 15 ℃;

diluting the solvent: n-hexane-isopropanol in a volume ratio of 95: 5;

mobile phase A: n-hexane-ethanol-diethylamine with a volume ratio of 99:1: 0.2;

mobile phase B: ethanol;

gradient elution conditions: as in example 1.

Preparing a test solution, a S- (-) -alpha-methylbenzyl isocyanate standard solution, a R- (+) -alpha-methylbenzyl isocyanate standard solution I, a R- (+) -alpha-methylbenzyl isocyanate standard solution II and a blank solution according to the determination method described in example 1, and recording chromatograms, wherein FIG. 6 is a high performance liquid chromatogram of the test solution.

As can be seen from FIG. 6, the peak-off time of S- (-) - α -methylbenzyl isocyanate was about 17.28 minutes, and the peak-off time of R- (+) - α -methylbenzyl isocyanate was about 16.51 minutes. The degrees of separation of S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate were 1.09.

Comparative example 1

The procedure of example 1 was followed except that the column was CHIRALPAK AD-H (4.6 x 250mm, 5 μm).

As shown in FIG. 7, S- (-) - α -methylbenzyl isocyanate and R- (+) - α -methylbenzyl isocyanate overlapped together in the peak and could not be separated efficiently.

Comparative example 2

The procedure was as in example 1 except that the diluting solvents were n-hexane and ethanol in a volume ratio of 90: 10.

The peak shapes of S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate are not good, so that the content of R- (+) -alpha-methylbenzyl isocyanate cannot be accurately calculated. And, the chromatographic peak separation degree is less than 1.

Comparative example 3

The procedure was as in example 1 except that the volume ratio of n-hexane to isopropyl alcohol was 99: 1.

The results show that the chromatographic peak separation degree of S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate is less than 1.

Comparative example 4

The procedure of example 1 was followed, except that the mobile phase a: n-hexane-ethanol-trifluoroacetic acid in a volume ratio of 90:10: 0.05.

The results show that the peaks of S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate overlap and the two substances cannot be separated efficiently.

Comparative example 5

The method of example 1 was followed except that the mobile phase A had a volume ratio of n-hexane, ethanol and diethylamine of 80:20: 0.05.

The results show that the chromatographic peak separation degree of S- (-) -alpha-methylbenzyl isocyanate and R- (+) -alpha-methylbenzyl isocyanate is less than 1.

Example 4

In order to further verify the effects of the detection method provided by the invention in all aspects, the following experiments are carried out:

1. specificity

According to the method of example 1, 10. mu.L each of a blank solvent (diluting solvent), a test sample solution, an S- (-) -alpha-methylbenzyl isocyanate standard solution and an R- (+) -alpha-methylbenzyl isocyanate standard two-solution was injected.

The experimental results show that: under the detection method, the blank solvent has no interference to the detection of the S- (-) -alpha-methylbenzyl isocyanate standard substance solution and the R- (+) -alpha-methylbenzyl isocyanate standard substance solution, and the detection method is proved to have strong specificity.

2. Precision of the system

Taking 10 mu L of the R- (+) -alpha-methylbenzyl isocyanate standard substance two solution in the example 1, continuously injecting a sample for 6 needles, recording a chromatogram, and determining peak areas, wherein the results are shown in the table 1.

TABLE 1 Peak-out time and Peak-out area

No.	1	2	3	4	5	6	Mean value	RSD％
									RTmin	16.670	16.671	16.670	16.672	16.669	16.669	16.670	0.01
Peak area	1.053	1.100	1.065	1.068	1.066	1.072	1.071	1.47

The calculated RSD of the retention time of the R- (+) -alpha-methylbenzyl isocyanate standard product is 0.01%, and the RSD of the peak area is 1.470.67%, thus the detection method has good system precision.

3. Linear experiment

20 mu L, 40 mu L, 50 mu L, 80 mu L and 100 mu L of the R- (+) -alpha-methylbenzyl isocyanate standard substance stock solution in the example 1 are respectively put into 5 measuring bottles with 20ml, and the diluted solvent in the example 1 is added to the measuring bottles to fix the volume and shake the measuring bottles evenly. The results of the peak areas of the samples injected separately are shown in Table 2 below:

TABLE 2 Peak area results

The linear equation is that y is 4.1779x-0.0057, and the correlation coefficient R²The result shows that the concentration and the peak area have good linear relation when the concentration of the R- (+) -alpha-methylbenzyl isocyanate is in the range of 0.103-0.513 mu g/ml.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.