阅读说明：本技术 一种测定环庚三烯肽类驱虫药中间体的分析方法 (Analysis method for determining cycloheptatriene peptide anthelmintic intermediate ) 是由 周莹 易中宏 吴禄春 张有理 于 2020-05-19 设计创作，主要内容包括：本发明为一种高效液相色谱法测定艾默德斯中间体1的纯度和杂质的方法,涉及医药技术领域。其技术方案为样品用适当方法制备成供试品溶液,以甲醇-四氢呋喃-水为流动相,在五氟苯基键合硅胶柱上将主成分艾默德斯中间体1和有关的杂质进行分离,用紫外检测器进行检测。此方法可有效地对艾默德斯中间体1的异构体进行分离,提高了艾默德斯工艺优化的效率。(The invention discloses a method for determining the purity and impurities of an Emerss intermediate 1 by using a high performance liquid chromatography, and relates to the technical field of medicines. The technical scheme is that a sample is prepared into a test solution by a proper method, methanol-tetrahydrofuran-water is used as a mobile phase, the main component of the Emerss intermediate 1 and related impurities are separated on a pentafluorophenyl bonded silica gel column, and an ultraviolet detector is used for detecting. The method can effectively separate the isomers of the Emerss intermediate 1, and improves the optimization efficiency of the Emerss process.)

1. A method for determining the purity and impurities of an emerald intermediate 1, characterized by: the chromatographic column is a chromatographic column taking pentafluoro-phenyl silane bonded silica gel as a filler; the mobile phase is a mixture of an organic solvent and water, and the detector is an ultraviolet detector.

2. The method of claim 1, wherein the chromatographic column has a column length of 250mm, a column inner diameter of 4.6mm and a particle size of 5 μm.

3. The method according to claims 1-2, wherein the volume ratio of the organic solvent to the purified water is 85:15 to 55: 45.

4. The method according to claim 3, wherein the organic solvent is a mixed solution of methanol and tetrahydrofuran.

5. The method according to claim 4, wherein the volume ratio of methanol to tetrahydrofuran is 11:9 to 13: 7.

6. The method of claim 5, wherein the detection wavelength is 200 to 230nm, 260 to 280 nm.

7. The method according to claim 6, wherein the column temperature of the chromatography column is from room temperature to 40 ℃.

8. The method of claim 7, wherein the flow rate of the chromatographic column is from 0.5 ml/min to 2.0 ml/min.

9. The method according to claim 8, wherein the concentration of the sample solution is 0.5 to 2.0 mg/ml.

10. The method according to claim 9, wherein the sample amount of the sample solution is 5 to 50 ul.

Technical Field

The invention belongs to the technical field of medical analysis, and particularly relates to an analysis method for determining an Emerss intermediate 1.

Background

Emerss (PF 1022-221) is a cycloheptatriene peptide with broad spectrum anthelmintic activity. The structural formula is as follows:

emersteus intermediate 1 is an intermediate produced in the first step of Emersteus synthesis and is obtained by nitration of PF 1022A. The structure is as follows:

the reaction system of the nitration process is quite complex, and the product has regioselective products except for the intermediate which is not completely converted in the nitro reduction process. The regioselective products of the nitration reaction are defined in patent WO2019040585a1 and are mainly: p-p, p-m, m-m, o-m products of two benzene rings of PF 1022A. The structure is as follows:

p-p

p-m

m-m

o-m

the process of obtaining the Emersproceeds intermediate 1 by the nitration of PF1022A is a key process for improving the yield of finished products, and p-p as a target product cannot be effectively separated from p-m by the existing method. Therefore, the realization of the separation of the isomer of the Emerss intermediate 1 is of great significance for optimizing the synthesis process of the Emerss and improving the yield of finished products.

Because the general alkyl bonded silica gel chromatographic column has limited separation effect on isomers, the chromatographic analysis is carried out on the Emersteus intermediate 1 by adopting a fluoro stationary phase which has stronger separation characteristics of ion exchange and polar action than an alkyl stationary phase and selecting a mixed solution of methanol and tetrahydrofuran water as a mobile phase.

Disclosure of Invention

The invention aims to provide a high performance liquid chromatography for separating and measuring an Emersoni intermediate 1, thereby realizing the quality control of the Emersoni intermediate 1.

The invention relates to a method for determining the purity and impurities of an Emersteur intermediate 1, which comprises the steps of injecting a sample solution of the Emersteur intermediate 1 into a chromatograph of a pentafluorophenylsilane bonded silica gel column, and separating and determining the purity of the Emersteur intermediate 1 or the related impurities, wherein a mobile phase is a mixture of an organic solvent and water, and a detector is an ultraviolet detector.

The preferred chromatographic column of the method of the present invention may be a chromatographic column using pentafluorophenylsilane bonded silica gel as a stationary phase, preferably a chromatographic column having a column length of 250mm, a column inner diameter of 4.6mm and a particle size of 5 μm, more preferably YuehuPFP 250mm × 4.6mm, 5um column.

In the mobile phase, the volume ratio of the organic solvent to the purified water is 85: 15-55: 45, preferably 70: 30; the organic solvent is a mixed solution of methanol and tetrahydrofuran, the volume ratio of the methanol to the tetrahydrofuran is 11: 9-13: 7, and the preferred volume ratio is 3: 2.

The ultraviolet detection wavelength of the invention is 200-230 nm, 260-280 nm, preferably 210nm, 270 nm.

The method of the invention further comprises the following steps:

(1) the column temperature of the chromatographic column is between room temperature and 40 ℃, and 40 ℃ is preferred;

(2) the flow rate of the chromatographic column is 0.5ml to 2.0ml/min, preferably 1.0 ml/min;

(3) the concentration of the test solution is 0.5-2.0 mg/ml, preferably 1.0 mg/ml;

(4) the sample amount of the test solution is 5-50 ul, preferably 10 ul.

The invention prepares a sample solution by a proper method, takes methanol-tetrahydrofuran-water as a mobile phase, separates the main component of the Emerskos intermediate 1 and related impurities on a pentafluorophenyl bonded silica gel column, and detects the product by an ultraviolet detector. The method can effectively separate the isomers of the Emerss intermediate 1, and improves the optimization efficiency of the Emerss process.

Drawings

FIG. 1 and the HPLC chart of example 1 show the chromatogram generated by the test sample on a phenyl-bonded silica gel chromatographic column, wherein chromatographic peak 1 is the coincidence peak of two sets of chromatographic peaks p-p and p-m, chromatographic peak 2 is the coincidence peak of two sets of chromatographic peaks m-m and m-o, and the resolution of chromatographic peaks 1 and 2 is 1.2.

FIG. 2 and the HPLC chart of example 2 show the chromatogram generated by the sample on the octyl-bonded silica gel column, wherein chromatographic peak 1 is the coincidence peak of two sets of chromatographic peaks of p-p and p-m, chromatographic peak 2 is the coincidence peak of two sets of chromatographic peaks of m-m and m-o, and the resolution of chromatographic peaks 1 and 2 is 2.1.

FIG. 3 and the HPLC chart of example 3 show the chromatogram generated by octadecyl bonded silica gel chromatography column of the sample, wherein chromatographic peak 1 is the coincidence peak of two groups of chromatographic peaks of p-p and p-m, chromatographic peak 2 is the coincidence peak of two groups of chromatographic peaks of m-m and m-o, and the resolution of chromatographic peaks 1 and 2 is 3.4.

FIG. 4 and the HPLC chart of example 4 show the chromatogram generated by the sample on a pentafluorophenyl bonded silica gel chromatographic column, wherein peak 1 is the peak of p-p, peak 2 is the coincidence peak of three groups of peaks p-m, m-m and m-o, and the resolution of peaks 1 and 2 is 1.1.

FIG. 5 and the HPLC chart of example 5 show the chromatogram condition of a sample in a 0.1% aqueous phosphoric acid solution and methanol as mobile phases, wherein peak 3 is a peak p-p chromatogram, peak 2 is a peak p-m chromatogram, peak 1 is a coincidence peak of two sets of peak m-m and m-o chromatogram, the degrees of separation of peaks 1 and 2 are 0.57, and the degrees of separation of peaks 2 and 3 are 0.85.

FIG. 6 and the HPLC chart of example 6 show the chromatogram condition of a sample in a 0.1% aqueous phosphoric acid solution and methanol as mobile phases, in which chromatogram peak 1 is a p-p chromatogram peak, chromatogram peak 2 is a p-m chromatogram peak, chromatogram peak 3 is a coincidence peak of two sets of chromatogram peaks m-m and m-o chromatogram peaks, the degrees of separation of chromatogram peaks 1 and 2 are 1.2, and the degrees of separation of chromatogram peaks 2 and 3 are 0.72.

FIG. 7 and the HPLC chart of example 7 show the chromatogram condition of a sample in a mobile phase of 0.1% phosphoric acid aqueous solution, methanol and acetonitrile, wherein chromatographic peak 1 is a chromatographic peak of p-p, chromatographic peak 2 is a coincidence peak of three chromatographic peaks of p-m, m-m and m-o, and the resolution of chromatographic peaks 1 and 2 is 1.3.

FIG. 8 and the HPLC chart of example 8 show the chromatogram of a sample under the conditions of a mobile phase of 0.1% phosphoric acid aqueous solution, tetrahydrofuran and acetonitrile, in which chromatogram peak 1 is a p-p chromatogram peak, chromatogram peak 2 is a p-m chromatogram peak, chromatogram peak 3 is a coincidence peak of two sets of chromatogram peaks of m-m and m-o chromatogram peaks, the degrees of separation of chromatogram peaks 1 and 2 are 0.81, and the degrees of separation of chromatogram peaks 2 and 3 are 0.69.

FIG. 9 and the HPLC chart of example 9 show the chromatogram condition of a sample in a mobile phase of 0.1% phosphoric acid aqueous solution, tetrahydrofuran and methanol, in which chromatogram peak 1 is a p-p chromatogram peak, chromatogram peak 2 is a p-m chromatogram peak, chromatogram peak 3 is a m-m chromatogram peak, chromatogram peak 4 is a m-m chromatogram peak, the degrees of separation of chromatogram peaks 1 and 2 are 1.6, the degrees of separation of chromatogram peaks 2 and 3 are 1.8, and the degrees of separation of chromatogram peaks 3 and 4 are 1.4.

Examples of the embodiments

Example 1

High performance liquid chromatograph: LC-2030C

A chromatographic column: phenyl silane bonded silica gel column (Agilent ZORBAX SB-Phenyl 4.6X 75mm 3.5um)

Mobile phase: 0.1% phosphoric acid water solution is used as a mobile phase A; acetonitrile as mobile phase B

The elution procedure for mobile phase A, B was:

flow rate: 1.0ml/min

Wavelength: 210nm

Sample introduction amount: 10ul of

Column temperature: 40 deg.C

The implementation steps are as follows:

test solution: preparing the sample of Emerss intermediate 1 into a solution having a concentration of about 1.0mg/ml

And (4) injecting 10 mu l of the test solution into a liquid chromatograph, and recording the chromatogram. The results are shown in FIG. 1.

Example 2

High performance liquid chromatograph: LC-2030C

A chromatographic column: octylsilane bonded silica gel column (Agilent ZORBAX SB-C84.6X 150mm 3.5um)

The same flows as in example 1;

the elution procedure for mobile phase A, B was:

the flow rate, wavelength, amount of sample introduction and column temperature were the same as in example 1

The implementation steps are as follows:

10. mu.l of the sample solution obtained in example 1 was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in FIG. 2.

Example 3

High performance liquid chromatograph: LC-2030C

A chromatographic column: octadecylsilane chemically bonded silica gel column (C18，4.6mm×250mm，5μm)

The same flows as in example 1;

the elution procedure for mobile phase A, B was:

the flow rate, wavelength, amount of sample introduction and column temperature were the same as in example 1

The implementation steps are as follows:

10. mu.l of the sample solution obtained in example 1 was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in FIG. 3.

Example 4

High performance liquid chromatograph: agilent1260

A chromatographic column: pentafluorophenylsilane bonded silica gel column (PFP，4.6mm×250mm，5μm)

The same flows as in example 1;

the elution procedure for mobile phase A, B was:

the flow rate, wavelength, amount of sample introduction and column temperature were the same as in example 1

The implementation steps are as follows:

10. mu.l of the sample solution obtained in example 1 was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in FIG. 4.

Example 5

High performance liquid chromatograph: agilent1260

Column chromatography as in example 4

Mobile phase: 0.1% phosphoric acid water solution is used as a mobile phase A; methanol is used as mobile phase B

The elution procedure for mobile phase A, B was:

the flow rate, sample introduction amount and column temperature were the same as in example 1

Wavelength: 270nm

The implementation steps are as follows:

10. mu.l of the sample solution obtained in example 1 was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in FIG. 5.

Example 6

High performance liquid chromatograph: agilent1260

Column chromatography as in example 4

Mobile phase: 0.1% phosphoric acid water solution is used as a mobile phase A; tetrahydrofuran as mobile phase B

The elution procedure for mobile phase A, B was:

the flow rate, wavelength, amount of sample introduction and column temperature were the same as in example 5

The implementation steps are as follows:

10. mu.l of the sample solution obtained in example 1 was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in FIG. 6.

Example 7

High performance liquid chromatograph: agilent1260

Column chromatography as in example 4

Mobile phase: water is a mobile phase A; methanol-acetonitrile (1:1) (v/v) as mobile phase B

The elution procedure for mobile phase A, B was:

the flow rate, wavelength, amount of sample introduction and column temperature were the same as in example 5

The implementation steps are as follows:

10. mu.l of the sample solution obtained in example 1 was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in FIG. 7.

Example 8

High performance liquid chromatograph: agilent1260

Column chromatography as in example 4

Mobile phase: water is a mobile phase A; tetrahydrofuran-acetonitrile (3:4) (v/v) as mobile phase B

The elution procedure for mobile phase A, B was:

the flow rate, wavelength, amount of sample introduction and column temperature were the same as in example 5

The implementation steps are as follows:

10. mu.l of the sample solution obtained in example 1 was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in FIG. 8.

Example 9

High performance liquid chromatograph: agilent1260

Column chromatography as in example 4

Mobile phase: water is a mobile phase A; tetrahydrofuran-methanol (2:3) (v/v) as mobile phase B

The elution procedure for mobile phase A, B was:

the flow rate, wavelength, amount of sample introduction and column temperature were the same as in example 5

The implementation steps are as follows:

10. mu.l of the sample solution obtained in example 1 was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in FIG. 9.