CN108362789B

CN108362789B - High performance liquid chromatography detection method for abamectin sodium optical isomer

Info

Publication number: CN108362789B
Application number: CN201810051439.5A
Authority: CN
Inventors: 柯兴斌; 左斌海; 赵红升; 张勇; 付长安
Original assignee: Zhuhai Yourun Co ltd
Current assignee: Zhuhai Yourun Co ltd
Priority date: 2018-01-19
Filing date: 2018-01-19
Publication date: 2020-09-01
Anticipated expiration: 2038-01-19
Also published as: CN108362789A

Abstract

The invention belongs to the technical field of drug analysis and detection, and particularly relates to a high performance liquid chromatography detection method of an abamectin sodium optical isomer. The method takes a xylonite IC column as a chromatographic column, a mobile phase is a mixture of ethanol, diethylamine and trifluoroacetic acid with the volume ratio of 100:0.15:0.1, the column temperature is 25-40 ℃, the flow rate is 1ml/min, and the detection wavelength is 210 nm. The invention has good specificity, sensitivity and reproducibility, and can accurately detect the content of the optical isomer in the avibactam sodium, thereby ensuring good clinical effect.

Description

High performance liquid chromatography detection method for abamectin sodium optical isomer

Technical Field

The invention belongs to the technical field of drug analysis and detection, and particularly relates to a high performance liquid chromatography detection method of an abamectin sodium optical isomer.

Background

Avibactam sodium (avibactam sodium), chemical name: [ (2S,5R) -2-acylamino-7-oxy-1, 6-diazabicyclo [3.2.1 ]]Octane-6-yl]Sodium sulfonate of the formula

The abamectin and the classical β -lactamase inhibitors (sulbactam, tazobactam and clavulanic acid) have essential difference in action mechanism, and in the process of inhibiting β -lactamase, the structure of the abamectin can be recovered through an inverse reaction, so that the abamectin has long-acting enzyme inhibition effect.

In the structure of the abamectin sodium, two chiral carbons exist, theoretically, four optical isomers exist, different optical isomers have large clinical activity difference, and some isomers even have strong adverse reactions. According to the SFDA chiral drug research guiding principle, the content of optical isomers of compounds with less than 3 chiralities cannot be controlled by using the chiral separation, and chiral chromatographic resolution control is required in principle to ensure the clinical medication safety. At present, no method for analyzing and detecting the abamectin sodium optical isomer exists.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a high performance liquid chromatography detection method of an abamectin sodium optical isomer. According to the invention, the xylonite IC column is selected as a chromatographic column, and the mobile phase is a mixture of ethanol, diethylamine and trifluoroacetic acid in a volume ratio of 100:0.15:0.1, so that the separation and detection of the avibactam sodium and the optical isomer thereof can be realized, and a reliable technical means is provided for the analysis of the avibactam sodium optical isomer.

The invention is realized by the following technical scheme:

a high performance liquid chromatography detection method of abamectin sodium optical isomer adopts a chromatographic column of xylonite IC (250 x 4.6mm 5 um); the mobile phase is alcohol: amine: a mixture of carboxylic acids in a volume ratio of 100:0.15: 0.1.

Preferably, the high performance liquid chromatography detection adopts the following chromatographic conditions:

the flow rate of the mobile phase is 0.5-2.0ml/min,

the temperature of the chromatographic column is 25-40 ℃,

the detector adopts an ultraviolet detector, and the detection wavelength is 200-230 nm.

Further preferably, the mobile phase flow rate is 1.0 ml/min; the temperature of the chromatographic column is 30 ℃; the detection wavelength of the detector is 210 nm.

Preferably, the stationary phase of the chromatographic column is silica gel with cellulose-tris (3, 5-dichlorophenyl carbamate) covalently bonded on the surface, the filler particle size is 2-10 μm, the column length is 100-250mm, and the column diameter is 2.1-4.6 mm.

Preferably, the alcohol is selected from one or a mixture of more than two of methanol, ethanol and propanol; the amine is selected from one of diethylamine and triethylamine; the acid is selected from one of formic acid, acetic acid and trifluoroacetic acid.

Further preferably, the mobile phase is prepared by mixing ethanol, diethylamine and trifluoroacetic acid in a volume ratio of 100:0.15:0.1 of a mixture.

High performance liquid chromatography is a separation technique widely used for chiral separation, and generally comprises normal phase chromatography and reverse phase chromatography, and separation of isomers in normal phase chromatography is superior to that in reverse phase system. Although the high performance liquid chromatography is widely applied to chiral separation, at present, the high performance liquid chromatography for separating the avibactam sodium and the optical isomer thereof is not reported. The inventor finds that the avibactam sodium has good solubility in water, but 4 optical isomers cannot be effectively separated by adopting chiral chromatographic columns (such as Daseluo IA, IB, IC and OZ-RH) with different stationary phases and selecting a reverse chromatographic system; and when a normal phase system of n-hexane/isopropanol, n-hexane/ethanol and n-hexane/methanol is adopted, the solubility of the abamectin sodium in the mobile phase is extremely low, and the detection requirement cannot be met.

The invention has the beneficial effects that:

according to the invention, the xylonite IC column is selected as a chromatographic column, the mobile phase is a mixture of ethanol, diethylamine and trifluoroacetic acid in a volume ratio of 100:0.15:0.1, the temperature of the chromatographic column is 25-40 ℃, the detection wavelength is 210nm, the separation and detection of the abamectin sodium and the optical isomer thereof can be realized, and a reliable technical means is provided for the analysis of the abamectin sodium and the optical isomer thereof. The invention has good specificity, sensitivity and reproducibility, and can accurately detect the content of the optical isomer in the avibactam sodium, thereby ensuring good clinical effect.

Drawings

FIG. 1 is a chromatogram of a resolution experiment;

FIG. 2 is a chromatogram of a sample testing experiment;

FIG. 3 is a chromatogram of an isomer experiment with a sample at a column temperature of 30 ℃;

FIG. 4 is a chromatogram of an isomer experiment with a sample at a column temperature of 40 ℃;

FIG. 5 is a chromatogram of a sample isomer experiment at 25 ℃.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1 separation degree test, sample test and sample application test

Instruments and reagents: shimadzu 2030C 3D high performance liquid chromatograph; ethanol was chromatographic grade, trifluoroacetic acid and diethylamine were analytical grade.

Sample preparation: avibactam sodium, its enantiomer (2R, 5S) avibactam sodium and 2 diastereomers.

The experimental method comprises the following steps:

chromatographic conditions are as follows: a column of xylonite IC (250 x 4.6mm 5 um); mixing the following raw materials in ethanol: diethylamine: trifluoroacetic acid (100:0.15:0.1) as a mobile phase at a flow rate of 1.0 ml/min; the column temperature is 30 ℃; the detector is an ultraviolet detector, and the detection wavelength is 210 nm.

Resolution experiments: respectively taking 10mg of abamectin sodium and enantiomer (2R, 5S) thereof, respectively, adding 20ml of ethanol for dissolving, and injecting 10 mu l of the solution into a liquid chromatograph, wherein the chromatogram is shown in figure 1.

Sample testing experiment: about 20mg of the practically produced abamectin sodium is taken and added with 20ml of ethanol for dissolution, 10 mu l of the solution is injected into a liquid chromatograph, and the chromatogram is shown in figure 2.

Sample addition isomer experiment: taking 5mg of each of 3 isomers of the abamectin sodium, adding 20ml of ethanol for dissolving, taking 1ml of the dissolved abamectin sodium, adding the dissolved abamectin sodium into a solution of a sample test experiment, and shaking up; 10. mu.l of the extract was injected into a liquid chromatograph, and the chromatogram was as shown in FIG. 3.

Experimental results and discussion:

the separation degree of the abamectin sodium and the enantiomer is 4.2; diastereomer 0.1% was detected in the sample; the isomers added into the sample can be effectively detected and well separated, which shows that the invention can effectively detect the content of the avibactam sodium isomer.

EXAMPLE 2 Effect of column temperature on separation

Sample preparation: avibactam sodium, enantiomer (2R, 5S) avibactam sodium and 2 diastereomers.

The experimental method comprises the following steps:

chromatographic conditions 1: a column of xylonite IC (250 x 4.6mm 5 um); mixing the following raw materials in ethanol: diethylamine: trifluoroacetic acid (100:0.15:0.1) as a mobile phase at a flow rate of 1.0 ml/min; the column temperature was 40 ℃; the detection wavelength was 210 nm.

Chromatographic conditions 2: a column of xylonite IC (250 x 4.6mm 5 um); mixing the following raw materials in ethanol: diethylamine: trifluoroacetic acid (100:0.15:0.1) as a mobile phase at a flow rate of 1.0 ml/min; the column temperature was 25 ℃; the detection wavelength was 210 nm.

Sample testing experiment: about 20mg of the practically produced abamectin sodium is taken and dissolved by adding 20ml of ethanol.

Sample addition isomer experiment: taking 5mg of each of 3 isomers of the abamectin sodium, adding 20ml of ethanol for dissolving, taking 1ml of the dissolved abamectin sodium, adding the dissolved abamectin sodium into a solution of a sample test experiment, and shaking up; 10. mu.l of the sample was taken and run under the chromatographic conditions 1 and 2, respectively, and the chromatograms obtained were as shown in FIGS. 4 and 5, respectively.

Experimental results and discussion:

the column temperature is 40 ℃, and the separation degree of the abamectin sodium and the enantiomer thereof is 3.8; the column temperature is 25 ℃, and the separation degree of the abamectin sodium and the enantiomer thereof is 4.0.

The common temperature has larger influence on chiral separation, the low temperature is favorable for chiral recognition, and the high temperature reduces the viscosity of a mobile phase and is favorable for mass transfer. The invention can effectively separate the avibactam sodium and the isomer thereof within the temperature range of 25-40 ℃.

Claims

1. A high performance liquid chromatography detection method of an abamectin sodium optical isomer is characterized in that a chromatographic column adopted by the method is a xylonite IC column with the thickness of 250 x 4.6mm and the thickness of 5 mu m; the mobile phase is ethanol: diethylamine: a mixture of trifluoroacetic acid in a volume ratio of 100:0.15: 0.1; the detector adopts an ultraviolet detector, and the detection wavelength is 200-230 nm.

2. The high performance liquid chromatography detection method of the abamectin sodium optical isomer according to claim 1, characterized in that the high performance liquid chromatography detection adopts the following chromatography conditions:

the flow rate of the mobile phase is 0.5-2.0ml/min,

the column temperature of the chromatographic column is 25-40 ℃.

3. The method for detecting the abamectin sodium optical isomer by high performance liquid chromatography according to claim 2, wherein the flow rate of the mobile phase is 1.0 ml/min; the temperature of the chromatographic column is 30 ℃; the detection wavelength of the detector is 210 nm.