An analytical method for determining intermediates of cycloheptatriene peptide anthelmintics

Abstract

The present invention relates to a method for determining the purity and impurities of Emendes intermediate 1 by high performance liquid chromatography, which belongs to the technical field of medicine. The technical solution is that the sample is prepared into a test solution by an appropriate method, methanol-tetrahydrofuran-water is used as the mobile phase, and the main component Emendes intermediate 1 and related impurities are separated on a pentafluorophenyl bonded silica column, and detected with a UV detector. This method can effectively separate the isomers of Emendes intermediate 1 and improve the efficiency of Emendes process optimization.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to an analytical method for determining the intermediate of cycloheptatriene peptide anthelmintics. Background Art

[0002] Emamectin (PF 1022-221) is a cycloheptatriene peptide with broad-spectrum anthelmintic activity. Its structural formula is as follows:

[0003]

[0004] Emamectin intermediate 1 is an intermediate produced in the first-step synthesis of emamectin, which is obtained by nitrating PF1022A. Its structure is as follows:

[0005]

[0006] The reaction system of the nitration process is very complex. In addition to the intermediate that is not completely converted during the nitro reduction process, the products also include regio-selective products. The regio-selective products of the nitration reaction are defined in Patent WO2019040585A1, mainly: the p-p, p-m, m-m, o-m products of the two benzene rings of PF1022A. Their structures are as follows:

[0007] p-p

[0008]

[0009] p-m

[0010]

[0011] m-m

[0012]

[0013] o-m

[0014]

[0015] The process of obtaining emamectin intermediate 1 by nitrating PF1022A is the key process to improve the yield of the finished product. As the target product, p-p cannot be effectively separated from p-m by the existing methods. Therefore, realizing the separation of the isomers of emamectin intermediate 1 is of great significance for optimizing the synthesis process of emamectin and improving the yield of the finished product.

[0016] Since the separation effect of general alkyl-bonded silica gel chromatographic columns on isomers is limited, a fluorinated stationary phase is used, which has stronger ion exchange and polar separation characteristics than the alkyl stationary phase. A mixed solution of methanol, tetrahydrofuran, and water is selected as the mobile phase for chromatographic analysis of emamectin intermediate 1. Summary of the Invention

[0017] The object of the present invention is to provide a high performance liquid chromatography method for separating and determining Emerdus intermediate 1, so as to realize the quality control of Emerdus intermediate 1.

[0018] The present invention is a method for determining the purity and impurities of Emerdus intermediate 1, which includes injecting a test solution of Emerdus intermediate 1 into a chromatograph with a pentafluorophenyl silane bonded silica gel column to separate and determine the purity of Emerdus intermediate 1 or its related impurities. Among them, the mobile phase is a mixture of an organic solvent and water, and the detector is an ultraviolet detector.

[0019] For the method described in the present invention, the preferred chromatographic column can be a chromatographic column with pentafluorophenyl silane bonded silica gel as the stationary phase, preferably a chromatographic column with a column length of 250 mm, a column inner diameter of 4.6 mm, and a particle size of 5 µm, and more preferably the Welch Ultimate® PFP 250 mm×4.6 mm, 5 µm chromatographic column.

[0020] In the mobile phase described in the present invention, the volume ratio of the organic solvent to purified water is 85:15 to 55:45, preferably 70:30; the organic solvent is a mixed solution of methanol and tetrahydrofuran, and the volume ratio of methanol to tetrahydrofuran is 11:9 to 13:7, preferably a volume ratio of 3:2.

[0021] The ultraviolet detection wavelength described in the present invention is 200 - 230 nm, 260 - 280 nm, preferably 210 nm, 270 nm.

[0022] The method described in the present invention further includes the following content:

[0023] (1) The column temperature of the chromatographic column is from room temperature to 40 °C, preferably 40 °C;

[0024] (2) The flow rate of the chromatographic column is 0.5 ml - 2.0 ml / min, preferably 1.0 ml / min;

[0025] (3) The concentration of the test solution is 0.5 - 2.0 mg / ml, preferably 1.0 mg / ml;

[0026] (4) The injection volume of the test solution is 5 - 50 μl, preferably 10 μl.

[0027] The present invention prepares a test solution by an appropriate method, uses methanol - tetrahydrofuran - water as the mobile phase, separates the main component Emerdus intermediate 1 and related impurities on a pentafluorophenyl silane bonded silica gel column, and detects with an ultraviolet detector. This method can effectively separate the isomers of Emerdus intermediate 1 and improve the efficiency of Emerdus process optimization. Description of the Drawings

[0028] Figure 1 The HPLC chromatogram of Example 1 is the chromatogram generated by the test sample on a phenyl-bonded silica gel chromatographic column. Chromatographic peak 1 is the overlapping peak of two groups of chromatographic peaks of p-p and p-m, chromatographic peak 2 is the overlapping peak of two groups of chromatographic peaks of m-m and m-o, and the resolution between chromatographic peaks 1 and 2 is 1.2.

[0029] Figure 2 The HPLC chromatogram of Example 2 is the chromatogram generated by the test sample on an octadecyl-bonded silica gel chromatographic column. Chromatographic peak 1 is the overlapping peak of two groups of chromatographic peaks of p-p and p-m, chromatographic peak 2 is the overlapping peak of two groups of chromatographic peaks of m-m and m-o, and the resolution between chromatographic peaks 1 and 2 is 2.1.

[0030] Figure 3 The HPLC chromatogram of Example 3 is the chromatogram generated by the test sample on an octadecyl-bonded silica gel chromatographic column. Chromatographic peak 1 is the overlapping peak of two groups of chromatographic peaks of p-p and p-m, chromatographic peak 2 is the overlapping peak of two groups of chromatographic peaks of m-m and m-o, and the resolution between chromatographic peaks 1 and 2 is 3.4.

[0031] Figure 4 The HPLC chromatogram of Example 4 is the chromatogram generated by the test sample on a pentafluorophenyl-bonded silica gel chromatographic column. Chromatographic peak 1 is the chromatographic peak of p-p, chromatographic peak 2 is the overlapping peak of three groups of chromatographic peaks of p-m, m-m and m-o, and the resolution between chromatographic peaks 1 and 2 is 1.1.

[0032] Figure 5 The HPLC chromatogram of Example 5 is the chromatogram generated under the chromatographic conditions where the mobile phase is 0.1% phosphoric acid aqueous solution and methanol. Chromatographic peak 3 is the chromatographic peak of p-p, chromatographic peak 2 is the chromatographic peak of p-m, chromatographic peak 1 is the overlapping peak of two groups of chromatographic peaks of m-m and m-o, the resolution between chromatographic peaks 1 and 2 is 0.57, and the resolution between chromatographic peaks 2 and 3 is 0.85.

[0033] Figure 6 The HPLC chromatogram of Example 6 is the chromatogram generated under the chromatographic conditions where the mobile phase is 0.1% phosphoric acid aqueous solution and methanol. Chromatographic peak 1 is the chromatographic peak of p-p, chromatographic peak 2 is the chromatographic peak of p-m, chromatographic peak 3 is the overlapping peak of two groups of chromatographic peaks of m-m and m-o, the resolution between chromatographic peaks 1 and 2 is 1.2, and the resolution between chromatographic peaks 2 and 3 is 0.72.

[0034] Figure 7 The HPLC chromatogram of Example 7 is the chromatogram generated under the chromatographic conditions where the mobile phase is 0.1% phosphoric acid aqueous solution, methanol and acetonitrile. Chromatographic peak 1 is the chromatographic peak of p-p, chromatographic peak 2 is the overlapping peak of three groups of chromatographic peaks of p-m, m-m and m-o, and the resolution between chromatographic peaks 1 and 2 is 1.3.

[0035] Figure 8 The HPLC chromatogram of Example 8 is the chromatogram generated by the test sample under the chromatographic conditions where the mobile phase is 0.1% phosphoric acid aqueous solution, tetrahydrofuran and acetonitrile. Chromatographic peak 1 is the chromatographic peak of p-p, chromatographic peak 2 is the chromatographic peak of p-m, chromatographic peak 3 is the overlapping peak of two groups of chromatographic peaks of m-m and m-o. The resolution between chromatographic peak 1 and 2 is 0.81, and the resolution between chromatographic peak 2 and 3 is 0.69.

[0036] Figure 9 The HPLC chromatogram of Example 9 is the chromatogram generated by the test sample under the chromatographic conditions where the mobile phase is 0.1% phosphoric acid aqueous solution, tetrahydrofuran and methanol. Chromatographic peak 1 is the chromatographic peak of p-p, chromatographic peak 2 is the chromatographic peak of p-m, chromatographic peak 3 is the chromatographic peak of m-m, chromatographic peak 4 is the chromatographic peak of m-m. The resolution between chromatographic peak 1 and 2 is 1.6, the resolution between chromatographic peak 2 and 3 is 1.8, and the resolution between chromatographic peak 3 and 4 is 1.4.

[0037] Implementation examples

[0038] Example 1

[0039] High performance liquid chromatograph: LC-2030C

[0040] Chromatographic column: Phenylsilane-bonded silica gel column (Agilent ZORBAX SB-Phenyl 4.6×75mm 3.5um)

[0041] Mobile phase: 0.1% phosphoric acid aqueous solution is mobile phase A; acetonitrile is mobile phase B

[0042] The elution program of mobile phase A and B is:

[0043]

[0044] Flow rate: 1.0 ml / min

[0045] Wavelength: 210 nm

[0046] Injection volume: 10 ul

[0047] Column temperature: 40 °C

[0048] Implementation steps:

[0049] Test sample solution: Take the test sample of Amides intermediate 1 and prepare a solution with a concentration of about 1.0 mg / ml.

[0050] Take 10 μl of the test sample solution and inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the appendix Figure 1 .

[0051] Example 2

[0052] High performance liquid chromatograph: LC-2030C

[0053] Chromatographic column: Octadecylsilyl silica gel column (Agilent ZORBAX SB-C8 4.6×150mm 3.5um)

[0054] The mobile phase is the same as that in Example 1;

[0055] The elution program of mobile phases A and B is as follows:

[0056]

[0057] The flow rate, wavelength, injection volume and column temperature are the same as those in Example 1

[0058] Implementation steps:

[0059] Take 10 μl of the test solution in Example 1 and inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the appendix Figure 2 .

[0060] Example 3

[0061] High performance liquid chromatograph: LC-2030C

[0062] Chromatographic column: Octadecylsilyl silica gel column (Ultimate ® C18, 4.6mm×250mm, 5 µm)

[0063] The mobile phase is the same as that in Example 1;

[0064] The elution program of mobile phases A and B is as follows:

[0065]

[0066] The flow rate, wavelength, injection volume and column temperature are the same as those in Example 1

[0067] Implementation steps:

[0068] Take 10 μl of the test solution in Example 1 and inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the appendix Figure 3 .

[0069] Example 4

[0070] High performance liquid chromatograph: Agilent1260

[0071] Chromatographic column: Pentafluorophenylsilyl silica gel column (Ultimate ® PFP, 4.6mm×250mm, 5 µm)

[0072] The mobile phase is the same as that in Example 1;

[0073] The elution procedures for mobile phases A and B are as follows:

[0074]

[0075] The flow rate, wavelength, injection volume, and column temperature are the same as in Example 1

[0076] Implementation steps:

[0077] Inject 10 μl of the test solution under Example 1 into the liquid chromatograph and record the chromatogram. The results are shown in the appendix Figure 4 .

[0078] Example 5

[0079] High-performance liquid chromatograph: Agilent1260

[0080] The chromatographic column is the same as in Example 4

[0081] Mobile phase: 0.1% phosphoric acid aqueous solution is mobile phase A; methanol is mobile phase B

[0082] The elution procedures for mobile phases A and B are as follows:

[0083]

[0084] The flow rate, injection volume, and column temperature are the same as in Example 1

[0085] Wavelength: 270 nm

[0086] Implementation steps:

[0087] Inject 10 μl of the test solution under Example 1 into the liquid chromatograph and record the chromatogram. The results are shown in the appendix Figure 5 .

[0088] Example 6

[0089] High-performance liquid chromatograph: Agilent1260

[0090] The chromatographic column is the same as in Example 4

[0091] Mobile phase: 0.1% phosphoric acid aqueous solution is mobile phase A; tetrahydrofuran is mobile phase B

[0092] The elution procedures for mobile phases A and B are as follows:

[0093]

[0094] The flow rate, wavelength, injection volume, and column temperature are the same as in Example 5

[0095] Implementation steps:

[0096] Inject 10 μl of the test solution under Example 1 into the liquid chromatograph and record the chromatogram. The results are shown in the appendix Figure 6。

[0097] Example 7

[0098] High performance liquid chromatograph: Agilent1260

[0099] The chromatographic column is the same as that in Example 4

[0100] Mobile phase: water is mobile phase A; methanol - acetonitrile (1:1) (v / v) is mobile phase B

[0101] The elution program of mobile phases A and B is as follows:

[0102]

[0103] The flow rate, wavelength, injection volume and column temperature are the same as those in Example 5

[0104] Implementation steps:

[0105] Take 10 μl of the test solution in Example 1 and inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the appendix Figure 7 。

[0106] Example 8

[0107] High performance liquid chromatograph: Agilent1260

[0108] The chromatographic column is the same as that in Example 4

[0109] Mobile phase: water is mobile phase A; tetrahydrofuran - acetonitrile (3:4) (v / v) is mobile phase B

[0110] The elution program of mobile phases A and B is as follows:

[0111]

[0112] The flow rate, wavelength, injection volume and column temperature are the same as those in Example 5

[0113] Implementation steps:

[0114] Take 10 μl of the test solution in Example 1 and inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the appendix Figure 8 。

[0115] Example 9

[0116] High performance liquid chromatograph: Agilent1260

[0117] The chromatographic column is the same as that in Example 4

[0118] Mobile phase: water is mobile phase A; tetrahydrofuran - methanol (2:3) (v / v) is mobile phase B

[0119] The elution program of mobile phases A and B is as follows:

[0120]

[0121] The flow rate, wavelength, sample injection volume and column temperature are the same as those in Example 5

[0122] Implementation steps:

[0123] Inject 10 μl of the test solution in Example 1 into the liquid chromatograph and record the chromatogram. The results are shown in the appendix Figure 9 .

Claims

1. A method for determining the purity and impurities of Amides intermediate 1, characterized in that: the chromatographic column is a chromatographic column filled with pentafluorophenyl silane-bonded silica gel; the detector is an ultraviolet detector; wherein, the structure of Amides intermediate 1 is as follows: ； the structures of the impurities are as follows: p-m 、 m-m 、 o-m ； mobile phase: water is mobile phase A; a mixed solution of tetrahydrofuran-methanol is mobile phase B; the volume ratio of the tetrahydrofuran to the methanol is 2:3; the elution program of mobile phases A and B is: 。 2. The method according to claim 1, wherein the chromatographic column is a chromatographic column with a column length of 250 mm, a column inner diameter of 4.6 mm, and a particle size of 5 µm.

3. The method according to claim 1 or 2, characterized in that the detection wavelength is 200-230 nm, 260-280 nm.

4. The method according to claim 3, characterized in that the column temperature of the chromatographic column is from room temperature to 40 °C.

5. The method according to claim 4, characterized in that the flow rate of the chromatographic column is 0.5 ml - 2.0 ml / min.

6. The method according to claim 5, characterized in that the concentration of the test solution is 0.5 - 2.0 mg / ml.

7. The method according to claim 6, characterized in that the injection volume of the test solution is 5 - 50 ul.

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