Method for detecting purity of eicosanedioic acid mono-tert-butyl ester

Abstract

The invention discloses a method for detecting the purity of mono-tert-butyl eicosanedioate, and mainly solves the technical problems of ultraviolet absorption intensity difference between a main product and known impurities, peak separation degree of adjacent known impurities and the like in the existing liquid chromatography method. The determination method comprises the following steps: 1, preparing a measurement reagent, 2, preparing a sample solution, and 3, carrying out high performance liquid chromatography determination, wherein a chromatographic instrument is a Siezanfo U3000 chromatographic instrument, an electric fog detector (CAD) is adopted, a chromatographic column is Shim-pack GLS CN from the Shimadjin company, and mobile phases are a TFA aqueous solution (A) with a mass percentage concentration of 0.1% and TFA acetonitrile (B) with a mass percentage concentration of 100%. The invention provides a reliable and convenient detection means for the method for detecting the purity of the mono-tert-butyl eicosanedioate.

Description

Technical Field

[0001] The present invention relates to a method for detecting the purity of tert-butyl esters, and particularly to a method for detecting the purity of monoterbutyl eicosanedioate. Background Art

[0002] For the conventional method for detecting the purity of monoterbutyl eicosanedioate, the high performance liquid chromatography method is required. Usually, a C18 chromatographic column is used, and the mobile phase uses acetonitrile / water. Since the main product has strong ultraviolet absorption and the known impurities have weak absorption under ultraviolet light, the conventional HPLC purity detection does not truly reflect the chemical purity of the product, and the conventional eluent will show serious tailing, and the peak symmetry is poor, and the known impurities are not completely separated. Summary of the Invention

[0003] The present invention provides a method for detecting the purity of monoterbutyl eicosanedioate, which mainly solves the technical problems such as different ultraviolet absorptions, peak tailing, and the resolution between the target peak and the adjacent known impurity peak existing in the existing liquid chromatography method.

[0004] The present invention adopts the following technical solutions: the method for detecting the purity of monoterbutyl eicosanedioate includes the following steps: Step 1: Prepare the reagents for measurement: acetonitrile - chromatographically pure, ultrapure water, and chromatographically pure trifluoroacetic acid.

[0005] Step 2: Preparation of the sample solution: Weigh the sample and place it in a volumetric flask, add water, dissolve it by ultrasonic wave at room temperature, then make up the volume with acetonitrile, and finally filter the sample with a polypropylene organic filter membrane and then conduct HPLC analysis.

[0006] Step 3: Perform high performance liquid chromatography determination; the chromatographic conditions are that the sample chamber temperature is 40 °C, the injection volume of the automatic injector is 10 μL, the chromatographic column uses Shim-pack GLS CN of Shimadzu Corporation, the column temperature: 40 °C, the flow rate: 1.0 mL / min, the mobile phase A is an aqueous solution of 0.1% TFA by mass percentage; the mobile phase B is an acetonitrile solution of 100% TFA by mass percentage, and a gradient elution program is adopted.

[0007] The chromatograph used in the liquid chromatography determination is a Thermo Fisher U3000 chromatograph, and this chromatograph uses a CAD detector. The chromatographic column size is 4.6 mm × 250 mm, 5.0 μm. The gradient elution program is as follows: 0 - 25 min, the volume ratio of the mobile phase B is from 60% to 100%, 25 - 25.1 min, the volume ratio of the mobile phase B changes from 100% to the initial ratio of 60%, 25.1 - 30 min, the volume ratio of the mobile phase B changes from 60% to balance the system.

[0008] The present invention has the following beneficial effects: In the research of the present invention, it is found that monoterbutyl eicosanedioate passes through a CAD detector, which is simple, fast, accurate, and has good stability. The corresponding values of the main product and known impurities through an electrospray detector also have a certain similarity; at the same time, the Shim-pack GLS CN chromatographic column of Shimadzu Corporation can provide sufficient hydrophobicity and retention ability, and it is found that it has good selectivity for compounds containing a cyano group, and well limits the diffusion distance in terms of adsorption and separation performance, solving the problems of peak tailing and the resolution of adjacent known impurity peaks. It achieves the technical effects of low concentration, high response, and baseline-level separation. Brief Description of the Drawings

[0009] Figure 1 HPLC chromatogram of the product. Detailed Embodiments

[0010] Main Instruments: Thermo Fisher U3000 high performance liquid chromatograph, equipped with a CAD detector, Shimadzu Shim-pack GLS CN chromatographic column (4.6 mm × 250 mm, 5.0 μm); ultrasonic vibrator (KQ5200E, Zhengzhou Kete Experimental Equipment Co., Ltd.), 0.22 μm polypropylene organic filter membrane (Shanghai Anpu Scientific Instruments Co., Ltd.), electronic analytical balance (CPA225D, Sartorius Scientific Instruments (Shanghai) Co., Ltd.).

[0011] Main Reagents: Acetonitrile (chromatographically pure, Merck, Germany), trifluoroacetic acid (chromatographically pure, Shanghai Guoyao Co., Ltd.); ultrapure water (resistivity 18.2 MΩ·cm, Thermo Fisher Scientific, USA).

[0012] Preparation of the sample solution: Weigh the monoterbutyl eicosanedioate sample and place it in a 5 ml volumetric flask, add 3 ml of methanol, dissolve it by ultrasound at room temperature, then make up the volume with ultrapure water, and finally filter the sample with a polypropylene organic filter membrane for chiral detection.

[0013] Chromatographic Conditions: The temperature of the sample chamber is room temperature, the injection volume of the autoinjector is 10 μL, Shim-pack GLS CN, column temperature: 40 °C, flow rate: 1.0 mL / min, mobile phase A is an aqueous solution of 0.1% TFA by mass percentage; mobile phase B is an acetonitrile solution of 100% TFA by mass percentage, and a gradient elution program is adopted. The gradient elution program is as follows: 0 - 25 min, the volume ratio of mobile phase B is 60% to 100%, 25 - 25.1 min, the volume ratio of mobile phase B changes from 100% to the initial ratio of 60%, 25.1 - 30 min, the volume ratio of mobile phase B changes from 60% to balance the system.

[0014] Selection and Optimization of Sample Pretreatment Method Currently, monoterbutyl docosanedioate in the market is a powdery solid, and the sample determined in this invention is the product produced by Gil Biochemical (Shanghai) Co., Ltd.

[0015] Optimization of Chromatographic Conditions In the selection of this invention, the separation effects were compared when the mobile phases were respectively aqueous solution of 0.1% TFA by mass - acetonitrile of 100% TFA by mass and aqueous solution of 0.025% formic acid by mass - acetonitrile of 0.025% formic acid by mass. When the system of aqueous solution of 0.025% formic acid by mass - methanol of 0.025% formic acid by mass was used, it was found that the chromatographic peak shape was asymmetric and prone to tailing; when the system of aqueous solution of 0.1% TFA by mass - acetonitrile of 100% TFA by mass was used as the eluent system, the symmetry degree of the peak with monoterbutyl docosanedioate reached the standard, and both the resolution and the peak shape of the target compound were better than those of other eluent systems. Therefore, in this experiment, the aqueous solution of 0.1% TFA by mass - acetonitrile of 100% TFA was used as the eluent, and gradient elution was carried out for chromatographic separation.

[0016] The sample chamber temperature of this invention was set at room temperature, which was beneficial to the preservation of acetonitrile solvent in the sample chamber. The column oven temperature was set at 40°C in this invention.

[0017] Determination of Actual Sample: The HPLC spectrum of the product is as Figure 1 shown. The results of chemical purity detection of monoterbutyl docosanedioate product by this method indicate that this method is fast, accurate, highly sensitive, the main peak has a symmetric shape, is baseline - separated from adjacent known impurity peaks, has good stability, and has practical application value.

Claims

1. Detection method for the purity of monoterbutyl docosanedioate; characterized in that: It includes the following steps: Step 1: Prepare the reagents for measurement: acetonitrile - chromatographic grade, ultrapure water, chemical reagent - trifluoroacetic acid; Step 2: Preparation of the sample solution: Weigh the test sample and place it in a volumetric flask, add water, dissolve it by ultrasonic wave at room temperature, then add acetonitrile to make up the volume. Finally, filter the test sample with a polypropylene organic filter membrane and then conduct chiral detection; Step 3: Perform high - performance liquid chromatography determination: The chromatographic conditions are as follows: the temperature of the sample chamber is at room temperature, an auto - sampler is used, and the chromatographic column is: The chromatographic column is Shim - pack GLS CN from Shimadzu Corporation, column temperature: 40 °C, flow rate: 1.0 mL / min, mobile phase A is an aqueous solution of 0.1% TFA by mass percentage concentration, mobile phase B is an acetonitrile solution of 100% TFA by mass percentage concentration, and a gradient elution program is adopted.

2. The method for detecting the purity of monoterbutyl docosanedioate according to claim 1, wherein: The chromatograph used in the liquid chromatography determination is the Thermo Fisher U3000 chromatograph, and this instrument uses a CAD detector.

3. The purity detection method of monoterbutyl eicosanedioate according to claim 1, characterized in that: The size of the chromatographic column is 4.6mm × 250mm, 5.0μm.

4. The purity detection method of monoterbutyl eicosanedioate according to claim 1, wherein: The gradient elution program is as follows: from 0 to 25 min, the volume ratio of mobile phase B is from 60% to 100%; from 25 to 25.1 min, the volume ratio of mobile phase B changes from 100% to the initial ratio of 60%; from 25.1 to 30 min, the volume ratio of mobile phase B changes from 60% to balance the system.

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