A method for removing liraglutide precursor main peak residues in a chromatographic column

Abstract

The present application relates to a method for removing the main peak residue of liraglutide precursor in a chromatographic column, and the liquid chromatography conditions are as follows: equipment configuration: four-element low-pressure pump or two-element high-pressure pump equipped with flow path switching valve; chromatographic column: InertSil ODS-SP chromatographic column (4.6*250mm, 5um); column temperature: 35-40 DEG C; mobile phase C: sulfate buffer: acetonitrile (82:18), 8M NaOH is adjusted to pH 5.95-6.05; mobile phase D: 50% acetonitrile; flow rate: 1.0ml / min; detection wavelength: 214nm; injection mode: 60% mobile phase D and 40% mobile phase C, isocratic elution for 8min, 0.01M phosphate buffer is injected, 30ul / needle, 1 needle is injected every 8min, a total of 3 needles. The analysis method of the present application can completely remove the main peak residue of liraglutide precursor in the chromatographic column in a short time.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pharmaceutical analysis chemistry, and particularly relates to a method for removing main peak residues of liraglutide precursor in a chromatographic column. BACKGROUND

[0002] Diabetes is a common metabolic endocrine disease, which has become the third chronic non-communicable disease threatening human health after cardiovascular and cerebrovascular diseases and malignant tumors. Diabetes is often accompanied by various complications, which is easy to cause damage to various organs, functional disorders and failure, and is very harmful.

[0003] Liraglutide is a GLP-1 analogue, which has the effects of reducing blood sugar, losing weight and benefiting cardiovascular diseases. As a subcutaneous injection preparation used once a week, liraglutide can play a good role in reducing blood sugar. Liraglutide precursor is a key intermediate in the production process of liraglutide, which is a polypeptide composed of 31 amino acids, and the molecular formula is: C 151 H 228 N 42 O 47 , and the molecular weight is 3383.7. The structure is as follows:

[0004] His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-

[0005] Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH

[0006] The detection method of liraglutide precursor is high performance liquid chromatography. When detected, the main peak of liraglutide precursor is easy to be left in the chromatographic column, and the residual amount signal-to-noise ratio (S / N) is 590.3 (see Figure 1 ), which affects the accurate quantification of subsequent liraglutide precursor samples. SUMMARY

[0007] In order to solve the problem that the main peak of liraglutide precursor is left in the analysis and detection chromatographic column, the application provides a method for removing the main peak residues of liraglutide precursor in the chromatographic column.

[0008] The technical scheme of the application is as follows:

[0009] A method for removing the main peak residues of liraglutide precursor in the chromatographic column, which adopts high performance liquid chromatography, and the high performance liquid chromatography conditions are as follows:

[0010] Chromatographic column: C18 chromatographic column;

[0011] Column temperature: 35-40 DEG C;

[0012] Mobile phase C: Sulfate buffer: Acetonitrile (volume ratio 82:18);

[0013] Mobile phase D: 50% acetonitrile (volume percent);

[0014] Flow rate: 1.0 ml / min;

[0015] Detection wavelength: 214 nm;

[0016] Further, the elution mode is: 60% mobile phase D and 40% mobile phase C, isocratic elution for 8 min;

[0017] Injection 0.01M phosphate buffer, 30 ul / needle, 1 needle every 8 min, a total of 3 needles. Remove the liraglutide precursor main peak residue.

[0018] Further, the pH of the mobile phase C is between 5.95~6.05.

[0019] Further, the chromatographic column is a C18 chromatographic column bonded with silica gel as the matrix.

[0020] Further, the inner diameter of the chromatographic column is 4.6 mm, the column length is 250 mm, and the filler particle size is 5 μm.

[0021] Further, the chromatographic column is an InertSil ODS-SP chromatographic column.

[0022] Compared with the prior art, the beneficial effects of the present application are:

[0023] The analysis method of the present application can make the liraglutide precursor main peak residue in the chromatographic column undetectable, and can effectively solve the problem of liraglutide precursor main peak residue in the analysis and detection chromatographic column. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Liraglutide precursor main peak residue chromatogram after the control solution

[0025] Figure 2 Liraglutide precursor main peak residue chromatogram for high performance liquid chromatograph for Shimadzu LC-20A (binary high pressure pump equipped with flow path switching valve) mobile phase C pH value is 5.95

[0026] Figure 3 Liraglutide precursor main peak residue chromatogram for high performance liquid chromatograph for Shimadzu LC-20A (binary high pressure pump equipped with flow path switching valve) mobile phase C pH value is 6.00

[0027] Figure 4LC-20A (binary high pressure pump equipped with flow path switching valve) for high performance liquid chromatograph, pH value of mobile phase C is 6.05, residual chromatogram of main peak of liraglutide precursor

[0028] Figure 5 Agilent 1260 (quaternary low pressure pump) for high performance liquid chromatograph, pH value of mobile phase C is 5.95, residual chromatogram of main peak of liraglutide precursor

[0029] Figure 6 Agilent 1260 (quaternary low pressure pump) for high performance liquid chromatograph, pH value of mobile phase C is 6.00, residual chromatogram of main peak of liraglutide precursor

[0030] Figure 7 Agilent 1260 (quaternary low pressure pump) for high performance liquid chromatograph, pH value of mobile phase C is 6.05, residual chromatogram of main peak of liraglutide precursor DETAILED DESCRIPTION

[0031] Instruments and reagents

[0032] Shimadzu LC-20A high performance liquid chromatograph was used, equipped with vacuum degassing machine, quaternary low pressure pump or binary high pressure pump equipped with flow path switching valve, automatic sampler, diode array (PDA) detector, and the control and recording of the chromatographic separation system were completed by Chromeleon 7 workstation.

[0033] Liraglutide precursor reference substance was provided by Jiangsu Wanbang Pharmaceutical Group Co., Ltd.; sodium dihydrogen phosphate was analytical pure (provided by Shanghai Chemical Reagent Co., Ltd. of China Pharmaceutical Group); anhydrous sodium sulfate was analytical pure (provided by Shanghai Chemical Reagent Co., Ltd. of China Pharmaceutical Group);

[0034] Ethanolamine was analytical pure (provided by Shanghai Chemical Reagent Co., Ltd. of China Pharmaceutical Group); sodium hydroxide was analytical pure (provided by Shanghai Chemical Reagent Co., Ltd. of China Pharmaceutical Group); acetonitrile was chromatographic grade (Merck KGaA); water was ultrapure water

[0035] The sample preparation method was as follows: about 10 mg of liraglutide precursor was accurately weighed and placed in a 10 ml volumetric flask, 0.01 mol / L phosphate buffer was added to dissolve and dilute to the mark, and a solution containing 1 mg of liraglutide precursor per 1 ml was prepared.

[0036] Liraglutide precursor main component peak detection method.

[0037] The high performance liquid chromatograph was Shimadzu LC-20A high performance liquid chromatograph or high performance liquid chromatograph Agilent LC-20A (quaternary low pressure pump).

[0038] Chromatographic column: InertSil® ODS-SP chromatographic column (4.6*250mm, 5μm);

[0039] Column temperature: 40°C;

[0040] Mobile phase A: Sulfate (sodium sulfate) buffer (pH 2.3): acetonitrile (volume ratio 82:18);

[0041] Mobile phase B: 50% acetonitrile; flow rate: 1.0 ml / min; detection wavelength: 214 nm;

[0042] Injection volume: 30ul;

[0043] Adjust the initial proportion of mobile phase (mobile phase A: mobile phase B = 55:45, volume ratio): make the liraglutide precursor main component peak out of the peak time about 20 min. Example

[0044] High performance liquid chromatograph is Shimadzu LC-20A (binary high pressure pump equipped with flow path switching valve)

[0045] Chromatographic column: InertSil® ODS-SP chromatographic column (4.6*250mm, 5μm);

[0046] Column temperature: 40°C;

[0047] Selection of mobile phase C:

[0048] Sulfate (sodium sulfate) buffer: acetonitrile (volume ratio 82:18), by adjusting the pH of mobile phase C to 5.95~6.05 with 8M NaOH, specifically, the pH of mobile phase C in this embodiment is 5.95, 6.00, 6.05 respectively.

[0049] Mobile phase D: 50% acetonitrile

[0050] Flow rate: 1.0 ml / min;

[0051] Detection wavelength: 214 nm;

[0052] Injection mode: according to the above liraglutide precursor main component peak detection method, after the liraglutide precursor main component is detected, 60% mobile phase D and 40% mobile phase C are used for isocratic elution for 8 min, 0.01M phosphate (sodium dihydrogen phosphate) buffer is injected, 30ul / needle, 1 needle every 8 min, a total of 3 needles, to remove the liraglutide precursor main peak residue. Replace with mobile phase AB, and determine the precursor main peak according to the liraglutide precursor main component peak detection method to determine the removal effect.

[0053] The results show that the pH of mobile phase C between 5.95 and 6.05 can effectively remove the residual of the main peak of liraglutide precursor in the chromatographic column (see Figures 2-4 ) ; it is shown that according to the method of Example 1, the residual of the main peak of liraglutide precursor in the chromatographic column is not detected. Example

[0054] The high performance liquid chromatograph is Agilent 1260 (four-element low-pressure pump)

[0055] The chromatographic column is InertSil OD S-SP chromatographic column (4.6*250mm, 5μm) ;

[0056] The column temperature is 40℃;

[0057] Selection of mobile phase C:

[0058] Sulfate (sodium sulfate) buffer: acetonitrile (volume ratio 82:18), by adjusting the pH of mobile phase C to 5.95~6.05 with 8M NaOH, specifically, the pH of mobile phase C in this example is 5.95, 6.00, 6.05 respectively.

[0059] Mobile phase D: 50% acetonitrile

[0060] Flow rate: 1.0ml / min;

[0061] Detection wavelength: 214nm;

[0062] Injection method: according to the above method for detecting the main component peak of liraglutide precursor, after the main component peak of liraglutide precursor is eluted, 60% mobile phase D and 40% mobile phase C are used for isocratic elution for 8min, 0.01M phosphate (sodium dihydrogen phosphate) buffer is injected, 30ul / needle, 1 needle every 8min, a total of 3 needles, to remove the residual of the main peak of liraglutide precursor. Replace with mobile phase AB, and determine the main peak of liraglutide precursor according to the method for detecting the main component peak of liraglutide precursor, to determine the removal effect.

[0063] The results show that the pH of mobile phase C between 5.95 and 6.05 can effectively remove the residual of the main peak of liraglutide precursor in the chromatographic column (see Figures 5-7 ) ; it is shown that according to the method of Example 2, the residual of the main peak of liraglutide precursor in the chromatographic column is not detected.

Claims

1. A method for removing a liraglutide precursor main peak remaining in a chromatographic column, characterized by, After the main component of liraglutide precursor is detected, the method for removing the residual liraglutide precursor main peak in the chromatographic column is performed, and the method is high performance liquid chromatography, and the high performance liquid chromatography conditions are as follows: The chromatographic column is a C18 chromatographic column. The column temperature is 35-40 DEG C. The mobile phase C is a sulfate buffer solution: acetonitrile, and the volume ratio is 82:18, and the pH of the mobile phase C is 5.95-6.

05. The mobile phase D is 50% acetonitrile. The flow rate is 1.0 ml / min. The detection wavelength is 214 nm. The elution mode is 60% mobile phase D and 40% mobile phase C, and isocratic elution for 8 min. The injection is 0.01M phosphate buffer solution, 30ul / needle, 1 needle every 8 min, and a total of 3 needles. The mobile phase A used for detecting the main component of liraglutide precursor is a pH2.3 sulfate buffer solution: acetonitrile, and the volume ratio is 82:18; the mobile phase B is 50% acetonitrile. The structure of liraglutide precursor is as follows: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH.

2. The method of claim 1, wherein, The chromatographic column is a C18 chromatographic column bonded with silica gel as a matrix.

3. The method of claim 1, wherein, The inner diameter of the chromatographic column is 4.6 mm, the column length is 250 mm, and the filler particle size is 5um.

4. The method of claim 1, wherein, The chromatographic column is an InertSil ODSP chromatographic column.

5. The method of claim 1, wherein, The column temperature is 40 DEG C.

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