Chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid

文档序号：466068 发布日期：2021-12-31 浏览：2次中文

阅读说明：本技术 2,3-二苯甲酰氧基-4-二甲基氨基-4-氧代丁酸的手性分离检测方法 (Chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid ) 是由陆茜刘青鲜孙雅丽于 2021-09-03 设计创作，主要内容包括：本发明提供了一种2,3-二苯甲酰氧基-4-二甲基氨基-4-氧代丁酸的手性分离检测方法,属于分析检测方法领域。本发明提供的分离检测方法,包括：包括如下步骤：使用稀释剂溶解待测样品,得待检测液；使用正相高效液相色谱法对待检测液进行检测,正相高效液相色谱法的色谱条件为：IC 4.6×250mm,5μm手性色谱柱,流动相由流动相A和流动相B组成,紫外检测波长为220nm-260nm,流速为0.5mL/min-1.5mL/min,柱温为25℃-35℃,流动相A为含有添加剂的乙醇,流动相B为正己烷,添加剂选自三乙胺、三乙醇胺以及甲酸中的任意一种或多种,洗脱程序为流动相A与流动相B的体积比为(60-70)：(30-40)。本发明能够简单有效地分离检测,而且峰型良好,分离度高。(The invention provides a chiral separation detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid, belonging to the field of analysis detection methods. The separation detection method provided by the invention comprises the following steps: the method comprises the following steps: dissolving a sample to be detected by using a diluent to obtain a liquid to be detected; detecting the liquid to be detected by using normal-phase high performance liquid chromatography, wherein the chromatographic conditions of the normal-phase high performance liquid chromatography are as follows: IC 4.6 is multiplied by 250mm,5 μm chiral chromatographic column, mobile phase is composed of mobile phase A and mobile phase B, ultraviolet detection wavelength is 220nm-260nm, flow rate is 0.5mL/min-1.5mL/min, column temperature is 25 ℃ -35 ℃, mobile phase A is ethanol containing additive, mobile phase B is n-hexane, additive is selected from one or more of triethylamine, triethanolamine and formic acid, elution procedure is that volume ratio of mobile phase A and mobile phase B is (60-70): (30-40). The invention can simply and effectively separate and detect, and has good peak shape and high separation degree.)

The chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid is characterized by comprising the following steps:

step 1, dissolving a sample to be detected by using a diluent to obtain a liquid to be detected;

step 2, detecting the liquid to be detected by using normal-phase high performance liquid chromatography,

wherein, the chromatographic conditions of the normal phase high performance liquid chromatography are as follows: IC 4.6 is multiplied by 250mm,5 mu m chiral chromatographic column, the mobile phase consists of a mobile phase A and a mobile phase B, the ultraviolet detection wavelength is 220nm-260nm, the flow rate is 0.5mL/min-1.5mL/min, the column temperature is 25 ℃ to 35 ℃,

the mobile phase A is ethanol containing additives, the mobile phase B is n-hexane,

the additive is one or more selected from triethylamine, triethanolamine and formic acid,

the elution procedure is that the volume ratio of the mobile phase A to the mobile phase B is (60-70): (30-40).

2. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 1, wherein:

wherein the diluent is any one or more of methanol, ethanol, acetonitrile and water.

3. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 2, wherein:

wherein the diluent is ethanol.

4. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 1, wherein:

wherein the concentration of the sample to be detected in the liquid to be detected is 5mg/mL-20 mg/mL.

5. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 1, wherein:

wherein the additive is 0.2-0.5 vol% triethylamine.

6. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 1, wherein:

wherein the additive is 0.2 vol% to 0.5 vol% triethanolamine and 0.2 vol% to 0.5 vol% formic acid.

7. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 5 or 6,

wherein the volume ratio of the mobile phase A to the mobile phase B in the elution procedure is 60: 40.

8. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 1,

wherein the flow rate is 0.5mL/min to 0.8 mL/min.

9. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 1,

wherein the detection wavelength is 230nm-245 nm.

10. The method for detecting chiral separation of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid according to claim 1,

wherein the chromatographic conditions further comprise:

the sample injection amount of the liquid to be detected is 1-5 mu L.

Technical Field

The invention relates to an analysis and detection method, in particular to a chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid.

Background

The structural formula of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid (hereinafter referred to as compound I) is:

the enantiomer of compound I is (2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid (hereinafter referred to as compound II), which has the structural formula:

in the prior art, a compound I and a compound II are generally used for forming salts with chiral amine, and chiral resolution is realized by utilizing the solubility difference of different chiral salts.

For example, chinese patent application No. 201880031547.4 discloses the use of compound I for resolution of racemic (cis) -3-acetamido-4-allyl-N- (tert-butyl) pyrrolidine-3-carboxamide, resulting in a product of single configuration in 77% yield and 99.5% ee value.

However, in the prior art, it is difficult to resolve and monitor the enantiomers of compound I and compound II by conventional methods.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide a highly efficient and accurate chiral separation and detection method for 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid.

The invention provides a chiral separation detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid, which is characterized by comprising the following steps: the method comprises the following steps: step 1, dissolving a sample to be detected by using a diluent to obtain a liquid to be detected; and 2, detecting the liquid to be detected by using a normal-phase high performance liquid chromatography, wherein the chromatographic conditions of the normal-phase high performance liquid chromatography are as follows: IC 4.6 is multiplied by 250mm,5 μm chiral chromatographic column, mobile phase is composed of mobile phase A and mobile phase B, ultraviolet detection wavelength is 220nm-260nm, flow rate is 0.5mL/min-1.5mL/min, column temperature is 25 ℃ -35 ℃, mobile phase A is ethanol containing additive, mobile phase B is n-hexane, additive is selected from one or more of triethylamine, triethanolamine and formic acid, elution procedure is that volume ratio of mobile phase A and mobile phase B is (60-70): (30-40).

In the chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid provided by the invention, the chiral separation and detection method can also have the following characteristics: wherein the diluent is one or more of methanol, ethanol, acetonitrile and water.

In the chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid provided by the invention, the chiral separation and detection method can also have the following characteristics: wherein the concentration of the sample to be detected in the liquid to be detected is 5mg/mL-20 mg/mL.

In the chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid provided by the invention, the chiral separation and detection method can also have the following characteristics: wherein the additive is triethylamine with the volume percent of 0.2-0.5.

In the chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid provided by the invention, the chiral separation and detection method can also have the following characteristics: wherein the additive is 0.2-0.5 vol% triethanolamine and 0.2-0.5 vol% formic acid.

In the chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid provided by the invention, the chiral separation and detection method can also have the following characteristics: wherein the volume ratio of the mobile phase A to the mobile phase B in the elution procedure is 60: 40.

In the chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid provided by the invention, the chiral separation and detection method can also have the following characteristics: wherein the chromatographic conditions further comprise: the sample volume of the solution to be detected is 1-5 muL.

Action and Effect of the invention

According to the chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid, a conventional chiral column and a proper mobile phase are selected, so that the method can be used for simply and effectively separating and detecting the compound and the enantiomer thereof, and is good in peak shape, high in resolution and suitable for quickly detecting the compound and the enantiomer thereof in an experiment.

Drawings

FIG. 1 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and an enantiomer thereof in example 1-1 of the present invention;

FIG. 2 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid in example 1-2 of the present invention;

FIG. 3 is a high performance liquid chromatogram of (2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid in example 1-2 of the present invention;

FIG. 4 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid in example 2 of the present invention;

FIG. 5 is a high performance liquid chromatogram of (2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid in example 2 of the present invention;

FIG. 6 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and its enantiomer in example 3 of the present invention;

FIG. 7 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and its enantiomer in example 4 of the present invention;

FIG. 8 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and enantiomer thereof in comparative example 1 of the present invention;

FIG. 9 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and its enantiomer in comparative example 2 of the present invention;

FIG. 10 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and an enantiomer thereof in comparative example 3 of the present invention;

FIG. 11 is a high performance liquid chromatogram of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and its enantiomer in comparative example 4 of the present invention.

Detailed Description

In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is specifically described below by combining the embodiment and the attached drawings.

In the following examples and comparative examples, the liquid phase apparatus used was a model 1100 high performance liquid chromatograph manufactured by Agilent.

In the following examples and comparative examples, the columns used were chiral columns manufactured by Daicel Chiralcel IC, Daicel Corp.

In the following examples and comparative examples, the proportions referred to in the mobile phase are volume ratios unless otherwise noted, and the proportions referred to in the elution gradient are volume ratios unless otherwise noted.

In the following examples and comparative examples, (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and (2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid were prepared by themselves, referring to Chinese patent application No. 201880031547.4.

In the following examples and comparative examples, a mixed sample of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid was obtained by mixing homemade (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid with homemade (2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid.

In the following examples, ethanol (containing 0.5% triethanolamine and 0.5% anhydrous formic acid) was prepared by adding 0.5 unit volume of triethanolamine and 0.5 unit volume of anhydrous formic acid to 100 unit volumes of ethanol and mixing them well, and a general preparation method was to add 0.5mL of triethanolamine and 0.5mL of anhydrous formic acid to 100mL of ethanol and mix them well.

In the following examples, ethanol (containing 0.2% triethylamine) is obtained by adding 0.2 unit volume of triethylamine to 100 unit volume of ethanol and mixing them uniformly, and a general preparation method may be to add 0.2mL of triethylamine to 100mL of ethanol and mix them uniformly.

< examples 1 to 1>

The embodiment provides a chiral separation detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid, which comprises the following specific steps:

step 1, diluting a 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid mixed sample by using ethanol to obtain a solution to be detected, wherein the concentration of the 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid mixed sample in the solution to be detected is 10 mg/mL.

And 2, detecting the liquid to be detected by using normal-phase high performance liquid chromatography, wherein the chromatographic conditions of the normal-phase high performance liquid chromatography are shown in table 1.

Table 1 table of liquid phase conditions of example 1

Chromatographic column	IC column (4.6X 250mm,5 μm)
		Mobile phase A	Ethanol (containing 0.5% triethanolamine and 0.5% anhydrous formic acid)
Mobile phase B	N-hexane
		Elution gradient	A-B(60:40)
Column temperature	35℃
		Flow rate of flow	0.8mL/min
Run time	25min
		Detection wavelength	230nm
Diluent	Ethanol
		Concentration of sample	10mg/mL
Sample volume	2μL
		Retention time	Compound IRT 9.942 min, compound IIRT 18.627 min

The liquid phase spectrum is shown in figure 1, a pair of enantiomers is completely separated in the spectrum, the base line is stable, the peak shape is good, and the peak appearance can be finished within 25 minutes.

< examples 1 and 2>

In this example, (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and (2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid were detected separately in the same manner as in example 1, and the liquid phase spectrum thereof is shown in FIG. 2-3, wherein the compound shown in FIG. 2 corresponds to (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid, and the compound shown in FIG. 3 corresponds to (2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid.

As shown in FIGS. 2-3, the peak position of the target compound has no interference of miscellaneous peaks, the baseline is smooth, and the peak pattern is good.

< example 2>

The embodiment provides a chiral separation detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid, which comprises the following specific steps:

step 1, diluting (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid or (2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid with ethanol to obtain a solution to be detected, wherein the concentration of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid or 2R, 3R) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid in the solution to be detected is 10 mg/mL.

Table 2 liquid phase conditions table for example 2

As shown in fig. 4-5, the liquid phase spectrum of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and its enantiomer under the liquid phase conditions provided in this example showed good separation, smooth baseline, and good peak shape, but the retention time was longer than that of example 1, but the peak was completed within 35 minutes.

< example 3>

The embodiment provides a chiral separation detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid, which comprises the following specific steps:

Table 3 table of liquid phase conditions of example 3

As shown in FIG. 6, the liquid phase spectrum of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and its enantiomer has better resolution and a smooth baseline, but the peak pattern is worse, when the liquid phase conditions provided in this example are used.

< example 4>

The embodiment provides a chiral separation detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid, which comprises the following specific steps:

Table 4 liquid phase conditions table for example 4

As shown in fig. 7, the liquid phase chromatogram is shown in fig. 7, when the liquid phase conditions provided in this example are adopted, the peaks of (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and the enantiomer thereof can be separated, the peak emergence is fast, the peak emergence can be completed within 15 minutes, but the separation degree is poor, and the baseline is not stable enough.

< comparative example 1>

The comparative example provides a separation detection method, comprising the following specific steps:

Table 5 liquid phase conditions table for comparative example 1

Chromatographic column	IC column (4.6 is multiplied by 250mm,5um)
		Mobile phase A	Ethanol
Mobile phase B	N-hexane
		Elution gradient	A-B(100:0)
Column temperature	35℃
		Flow rate of flow	0.5mL/min
Run time	30min
		Detection wavelength	230nm
Diluent	Ethanol
		Concentration of sample	10mg/mL
Sample volume	2μL

The liquid phase spectrum is shown in FIG. 8, and (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and the enantiomer thereof could not be separated under the liquid phase condition.

< comparative example 2>

The comparative example provides a separation detection method, comprising the following specific steps:

Table 6 liquid phase conditions table of comparative example 2

Chromatographic column	IC column (4.6 is multiplied by 250mm,5um)
		Mobile phase A	Ethanol
Mobile phase B	N-hexane
		Elution gradient	A-B(70:30)
Column temperature	35℃
		Flow rate of flow	0.5mL/min
Run time	20min
		Detection wavelength	230nm
Diluent	Ethanol
		Concentration of sample	10mg/mL
Sample volume	2μL

The liquid phase spectrum is shown in FIG. 8, and (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and its enantiomer cannot be completely separated under the liquid phase condition.

< comparative example 3>

The comparative example provides a separation detection method, comprising the following specific steps:

Table 7 liquid phase conditions table for comparative example 3

Chromatographic column	AD-H column (4.6X 250mm,5um)
		Mobile phase A	Ethanol
Mobile phase B	N-hexane
		Elution gradient	A-B(70:30)
Column temperature	35℃
		Flow rate of flow	0.5mL/min
Run time	20min
		Detection wavelength	230nm
Diluent	Ethanol
		Concentration of sample	10mg/ml
Sample volume	2μL

The liquid phase spectrum is shown in FIG. 9, and (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and the enantiomer thereof could not be completely separated under the liquid phase condition.

< comparative example 4>

The comparative example provides a separation detection method, comprising the following specific steps:

Table 8 liquid phase conditions table for comparative example 4

Chromatographic column	OD-H column (4.6X 250mm,5um)
		Mobile phase A	Ethanol
Mobile phase B	N-hexane
		Elution gradient	A-B(70:30)
Column temperature	35℃
		Flow rate of flow	0.5mL/min
Run time	30min
		Detection wavelength	230nm
Diluent	Ethanol
		Concentration of sample	10mg/mL
Sample volume	2μL

The liquid phase spectrum is shown in FIG. 9, and (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and the enantiomer thereof cannot be separated under the liquid phase condition, and the baseline is not smooth and is not suitable for use.

Effects and effects of the embodiments

According to the chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid, due to the addition of the appropriate additive in the mobile phase A, the (2S, 3S) -2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutanoic acid and the enantiomer thereof can be successfully and effectively separated, and particularly after 0.5% triethanolamine and 0.5% anhydrous formic acid are added in the mobile phase A, two peaks can be separated at high separation degree, the peak shape is good, and the baseline is stable.

The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.

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Chiral separation and detection method of 2, 3-dibenzoyloxy-4-dimethylamino-4-oxobutyric acid

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