Degarelix acetate impurity detection method

Abstract

The invention relates to a degarelix acetate impurity detection method, and belongs to the technical field of medicinal chemistry, and the method comprises the following steps: respectively dissolving a sample to be detected and an impurity standard substance with 0.1% trifluoroacetic acid-acetonitrile (volume ratio of 80: 20) to prepare a test solution and an impurity reference substance solution, adopting an octadecylsilane bonded silica gel chromatographic column, and detecting the content of degarelix acetate impurities in the sample to be detected. Trifluoroacetic acid-acetonitrile (mobile phase A) and acetonitrile (mobile phase B) containing a phosphate buffer system are used for gradient elution, and high performance liquid chromatography is carried out under the wavelength of 226nm. The degarelix acetate impurities can be specifically recognized and quantified through contrast chromatograms. The method has the advantages of high sensitivity, high separation efficiency and strong stability, and is especially suitable for quality control of impurities in degarelix acetate bulk drug production.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and particularly to a method for detecting impurities of degarelix acetate. Background Art

[0002] Degarelix Acetate (D-alaninamide, N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-seryl-4-[[[(4S)-hexahydro-2,6-dioxo-4-pyrimidinyl]carbonyl]amino]-L-phenylalanyl-4-[(carbamoyl)amino]-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-prolyl) is a synthetic decapeptide drug and belongs to the third-generation gonadotropin-releasing hormone (GnRH) receptor antagonist. Its molecular structure consists of 10 amino acids, and the core sequence is Ac-D-2-Nal-D-Phe-(4-Cl)-D-3-Pal-Ser-4-Aph(L-Hor)-D-4-Aph(Cbm)-Leu-Lys(ipr)-Pro-D-Ala-NH 2 , and its molecular formula is C 82 H 103 ClN 18 O 16 , and its molecular weight is 1632.26. This drug can rapidly reduce testosterone levels by reversibly inhibiting pituitary GnRH receptors and blocking endogenous gonadotropin release, and is used for the treatment of hormone-dependent prostate cancer.

[0003] In the prior art, the synthesis method of degarelix acetate is mainly based on solid-phase polypeptide synthesis technology, and the amino acid sequence is gradually coupled by using the Fmoc protection strategy. The main steps include resin pretreatment and deprotection, introduction and removal of side-chain protecting groups, cleavage and purification. Inevitably, a series of impurities are introduced during this process, including racemization impurities, misconnection impurities, and deletion peptide impurities, etc. The existence of these impurities may seriously affect the therapeutic effect of degarelix acetate. For example, structurally similar impurities (such as deletion peptide impurities) may interfere with the binding of degarelix acetate to GnRH receptors and weaken its anti-prostate cancer effect of inhibiting testosterone release, while non-natural configuration impurities (such as racemization impurities) may induce immune reactions and lead to a decrease in treatment tolerance. Therefore, in order to ensure the product quality of degarelix acetate and improve the drug safety of patients, it is of great practical significance to strictly control the related substances of degarelix acetate. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method for detecting impurities in degarelix acetate. A dissolving solution prepared with 0.1% trifluoroacetic acid and acetonitrile is used to dissolve the sample to be tested. An octadecylsilyl-bonded silica gel chromatographic column is employed, and gradient elution is carried out with trifluoroacetic acid-acetonitrile containing a phosphate buffer system. High-performance liquid chromatography analysis is performed at a wavelength of 226 nm. By comparing the chromatograms, the impurities in degarelix acetate can be specifically identified and quantified.

[0005] The object of the present invention is to provide a method for detecting impurities in degarelix acetate, and the detection method includes the following steps:

[0006] Step S1: Dissolve the sample to be tested with a trifluoroacetic acid-acetonitrile solution to obtain a test solution;

[0007] Step S2: Dissolve the impurity reference standard with a trichloroacetic acid-acetonitrile solution to obtain an impurity reference solution;

[0008] Step S3: Detect the test solution and the impurity reference solution respectively by high-performance liquid chromatography. By comparing and analyzing the chromatograms of the test solution and the impurity reference solution, the types and contents of the impurities in the sample to be tested are obtained. The high-performance liquid chromatography uses octadecylsilyl-bonded silica gel as the filler and a trifluoroacetic acid-acetonitrile system as the mobile phase.

[0009] Further, in Step S1 and Step S2, the concentration of trifluoroacetic acid in the trifluoroacetic acid-acetonitrile solution is 0.1%, and the volume ratio of trifluoroacetic acid to acetonitrile is 80:20.

[0010] Further, in Step S1, the concentration of the sample to be tested in the test solution is 1 mg / mL.

[0011] Further, in Step S1, the sequence structural formula of the impurity is one or more of the following:

[0012] (1)Ac-[D-2-Nal]-[D-4-Cpa]-[D-3-Pal]-Ser-[4-Aph(D-Hor)]-[D-4-Aph(Cbm)]-Leu-[Lys(ipr)]-Pro-[D-Ala]-NH 2 ;

[0013] (2)Ac-D-2-Nal-D-4-Cpa-3-Pal-Ser-4-Aph(L-Hor)-D-4-Aph(Cbm)-Leu-Lys(ipr)-Pro-D-Ala-NH 2 ;

[0014] (3) Ac-D-2-Nal-D-4-Cpa-D-3-Pal-Ser-4-Aph(L-Hor)-D-4-Aph(Cbm)-Leu-D-Lys(ipr)-Pro-D-Ala-NH 2 。

[0015] Further, in step S3, the pore size of the packing material of the high performance liquid chromatography is

[0016] Preferably, in step S3, the pore size of the packing material of the high performance liquid chromatography is

[0017] Further, in step S3, the particle size of the packing material of the high performance liquid chromatography is 3 - 5 μm.

[0018] Preferably, in step S3, the particle size of the packing material of the high performance liquid chromatography is 5 μm.

[0019] Further, in step S3, the packing material of the high performance liquid chromatography is a capped packing material.

[0020] In one embodiment of the present invention, the chromatographic column of the high performance liquid chromatography is YMC-Pack ODS AQ (4.6×250 mm, 5 μm).

[0021] In one embodiment of the present invention, the column temperature of the high performance liquid chromatography is 50 - 55 °C.

[0022] Further, in step S3, the high performance liquid chromatography uses an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 226 nm.

[0023] In one embodiment of the present invention, the injection volume of the high performance liquid chromatography is 20 μL.

[0024] Further, in step S3, the high performance liquid chromatography uses trifluoroacetic acid - acetonitrile solution as mobile phase A and acetonitrile as mobile phase B, and trifluoroacetic acid and acetonitrile in mobile phase A are mixed in a sodium dihydrogen phosphate - disodium hydrogen phosphate buffer system.

[0025] Further, in step S3, the high performance liquid chromatography uses gradient elution, and the conditions of the gradient elution are as follows:

[0026] 0 min, 60% volume ratio of mobile phase A and 30% volume ratio of mobile phase B;

[0027] 30 min, 40% volume ratio of mobile phase A and 60% volume ratio of mobile phase B;

[0028] 35 min, 70% volume ratio of mobile phase B;

[0029] 36 - 45 minutes, 60% by volume of mobile phase A and 30% by volume of mobile phase B.

[0030] Further, in step S3, the flow rate of the high - performance liquid chromatography mobile phase is 0.9 - 1 mL / min.

[0031] Advantages of the present invention:

[0032] The present invention can rapidly and efficiently detect the types and contents of degarelix acetate impurities in a sample to be tested by high - performance liquid chromatography. By screening and adjusting important parameters such as the dilution liquid system, mobile phase system, ultraviolet detection wavelength, and chromatographic column packing, a set of high - performance chromatographic detection methods suitable for the detection of degarelix acetate impurities is finally successfully obtained, which can be applied to the quality control of impurities in the synthesis process of degarelix acetate, and has the characteristics of high sensitivity, high separation efficiency, and strong stability. Description of the Drawings

[0033] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in conjunction with the drawings, where

[0034] Figure 1 is the ultraviolet spectrum result diagram of degarelix acetate and different impurities in Example 1 of the present invention;

[0035] Figure 2 is the chromatogram result diagram obtained by detecting and analyzing with mobile phase 1 in Example 3 of the present invention;

[0036] Figure 3 is the chromatogram result diagram obtained by detecting and analyzing with mobile phase 2 in Example 3 of the present invention;

[0037] Figure 4 is the chromatogram result diagram obtained by detecting and analyzing with mobile phase 3 in Example 3 of the present invention;

[0038] Figure 5 is the chromatogram result diagram obtained by detecting and analyzing with end - capped packing in Example 4 of the present invention;

[0039] Figure 6 is the chromatogram result diagram obtained by detecting and analyzing with non - end - capped packing in Example 4 of the present invention. Detailed Embodiments

[0040] The following further illustrates the present invention in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0041] The following impurities of degarelix acetate are involved in the following examples:

[0042] Racemic impurity I:

[0043] The sequence structural formula is Ac-[D-2-Nal]-[D-4-Cpa]-[D-3-Pal]-Ser-[4-Aph(D-Hor)]-[D-4-Aph(Cbm)]-Leu-[Lys(ipr)]-Pro-[D-Ala]-NH 2 , with a molecular weight of 1632.3;

[0044] Racemic impurity II:

[0045] The sequence structural formula is Ac-D-2-Nal-D-4-Cpa-3-Pal-Ser-4-Aph(L-Hor)-D-4-Aph(Cbm)-Leu-Lys(ipr)-Pro-D-Ala-NH 2 , with a molecular weight of 1632.3;

[0046] Racemic impurity III:

[0047] The sequence structural formula is Ac-D-2-Nal-D-4-Cpa-D-3-Pal-Ser-4-Aph(L-Hor)-D-4-Aph(Cbm)-Leu-D-Lys(ipr)-Pro-D-Ala-NH 2 , with a molecular weight of 1632.3.

[0048] Example 1: Selection of wavelength

[0049] The ultraviolet spectra of degarelix acetate and different impurities (including deletion peptide impurities, misconnection impurities and racemic impurities, including the above racemic impurity I, racemic impurity II and racemic impurity III) were extracted respectively. The results are as Figure 1 shown. The ultraviolet spectra of each impurity are similar to that of degarelix, and they all have a maximum absorption near 226 nm. Therefore, 226 nm is used as the detection wavelength for degarelix acetate impurities.

[0050] Example 2: Selection of diluent

[0051] Diluent 1: water;

[0052] Diluent 2: 50% acetonitrile;

[0053] Diluent 3: 0.1% trifluoroacetic acid - acetonitrile (volume ratio 80:20)

[0054] (1) Solubility test

[0055] Weigh approximately 10 mg of the degarelix acetate sample respectively, place it in a 10-mL volumetric flask, and add three diluents: water, 50% acetonitrile, and 0.1% trifluoroacetic acid-acetonitrile (80:20). Shake well. It was observed that the sample was completely dissolved in water, 50% acetonitrile, and 0.1% trifluoroacetic acid-acetonitrile (80:20), and the solution was clear and transparent without particles or precipitation. After further filtration through a 0.22-μm filter membrane, there was no residue on the filter membrane of the 0.1% trifluoroacetic acid-acetonitrile (80:20) diluent, indicating that this diluent has the best dissolving ability for the sample.

[0056] (2) Stability test

[0057] Weigh approximately 10 mg of the degarelix acetate sample respectively, place it in a 10-mL volumetric flask, and add three diluents: water, 50% acetonitrile, and 0.1% trifluoroacetic acid-acetonitrile (80:20). Shake well and investigate its stability at room temperature and refrigeration (refrigeration temperature is 2 - 8 °C). The results showed that when using 0.1% trifluoroacetic acid-acetonitrile (80:20) as the diluent, after the sample was placed at room temperature for 24 hours, the change rate of the main component peak area was less than 2%, the retention time had no obvious drift, and no new impurity peaks were observed. While using water and 50% acetonitrile as diluents, obvious degradation occurred after 12 hours, the change rate of the peak area was greater than 5%, and new impurity peaks appeared, indicating that the 0.1% trifluoroacetic acid-acetonitrile (80:20) diluent can effectively maintain the chemical stability of the sample.

[0058] (3) Peak shape test

[0059] Weigh approximately 10 mg of the degarelix acetate sample respectively, place it in a 10-mL volumetric flask, and add three diluents: water, 50% acetonitrile, and 0.1% trifluoroacetic acid-acetonitrile (80:20). Shake well and use HPLC method to inject and analyze the above three diluted sample solutions respectively. The results showed that when using 0.1% trifluoroacetic acid-acetonitrile as the diluent, the symmetry factor (As) of the main component peak shape was 1.05, the tailing factor (Tf) was 1.12, and the number of theoretical plates (N) was 12500. The peak shape was sharp and symmetric without tailing phenomenon. While in water and 50% acetonitrile, obvious tailing occurred in the peak shape (Tf > 1.8), and the symmetry was poor (As > 1.3), indicating that the 0.1% trifluoroacetic acid-acetonitrile diluent can provide the best chromatographic peak shape.

[0060] Based on the above three experimental results (see Table 1), using 0.1% trifluoroacetic acid-acetonitrile (80:20) as the diluent to dilute the sample can completely dissolve degarelix acetate, maintain the chemical stability of the sample at room temperature and refrigeration conditions, provide a symmetric and sharp chromatographic peak shape, and meet the requirements of the related substances analysis method.

[0061] Table 1 Results of diluent selection

[0062]

[0063] Example 3: Selection of mobile phase buffer salts

[0064] In the high performance liquid chromatography (HPLC) detection method, the selection of the mobile phase buffer salt system has an important impact on the chromatographic separation effect, peak shape and the accuracy of the analysis results. The traditional selection of the mobile phase mostly relies on experience or fixed formulas, which is difficult to meet the analysis requirements of complex samples and may lead to problems such as insufficient resolution, peak tailing or poor method stability. Therefore, a systematic method is needed to optimize the mobile phase buffer salt system to meet the analysis requirements of different samples.

[0065] This example is based on an optimization method and system for the mobile phase buffer salt system, which can automatically screen and optimize the mobile phase buffer salt system according to the sample characteristics and analysis objectives, improve the separation effect, analysis precision and method stability, so as to determine the best mobile buffer salt for detecting the impurities of degarelix acetate.

[0066] The buffer solution conditions (0.1% trifluoroacetic acid - acetonitrile (80:20)) screened in Example 2 were used to dilute the degarelix acetate samples containing Impurity I, Impurity II and Impurity III. The UV spectral conditions (226 nm) screened in Example 1 were combined with the following different mobile phase buffer solution systems as mobile phase A, and the diluted degarelix acetate sample solution was subjected to HPLC detection and analysis. The results are shown in Table 2.

[0067] Mobile phase 1: 0.05 mol / L potassium dihydrogen phosphate solution (pH = 2.5);

[0068] Mobile phase 2: 0.1% trifluoroacetic acid solution;

[0069] Mobile phase 3: Measure 920 mL of buffer solution [buffer solution: 0.025 mol / L sodium dihydrogen phosphate solution and 0.025 mol / L disodium hydrogen phosphate solution (pH = 2.5)], add 1 mL of trifluoroacetic acid, mix well, and then add 80 mL of acetonitrile.

[0070] Table 2 Results of buffer solution selection

[0071] Mobile phase Resolution of impurity I Resolution of impurity II Resolution of impurity III Theoretical plate number of main peak 1 Well separated, 4.03 Slightly better separated, 1.45 Poor peak shape of impurity III 131210 2 Well separated, 3.85 Not baseline separated Not baseline separated 147520 3 Well separated, 8.52 Well separated, 5.71 Well separated, 3.64 191026

[0072] Selecting the mobile phase 3 system as mobile phase A to detect the diluted degarelix acetate samples, the separation of Impurity I, Impurity II and Impurity III is very good, and the theoretical plate number of the main peak is the highest.

[0073] Example 4: Selection of chromatographic column

[0074] According to the polarities of the degarelix acetate sample, impurity I, impurity II, and impurity III, a chromatographic column filled with octadecylsilyl silica gel is selected.

[0075] According to the molecular weights of the degarelix acetate sample, impurity I, impurity II, and impurity III (all around 1600), the pore size range of the packing material is selected within

[0076] According to the separation requirements, the particle size of the packing material is selected. The smaller the particle size, the better the separation effect, but the higher the column pressure. Therefore, the particle size range of the packing material is selected to be 3 - 5 μm.

[0077] According to the polarities (medium polarities) of degarelix acetate, impurity I, impurity II, and impurity III, a chromatographic column with low-density bonding is selected. It has certain hydrophilicity and can better retain medium-polarity compounds.

[0078] The degarelix acetate sample containing impurity I, impurity II, and impurity III is diluted using the buffer conditions (0.1% trifluoroacetic acid - acetonitrile (80:20)) screened in Example 2. Using the ultraviolet spectral conditions (226 nm) screened in Example 1 and the mobile phase buffer system (measure 920 mL of buffer [buffer: 0.025 mol / L sodium dihydrogen phosphate solution and 0.025 mol / L disodium hydrogen phosphate solution (pH = 2.5)], add 1 mL of trifluoroacetic acid, mix well, and then add 80 mL of acetonitrile) screened in Example 3, the samples are detected and analyzed using either end-capped or non-end-capped packing materials. The chromatographic results using end-capped packing materials are as Figure 5 shown, and the chromatographic results using non-end-capped packing materials are as Figure 6 shown. It can be seen that by selecting end-capped packing materials, the tailing phenomenon of basic compounds can be reduced.

[0079] Example 5: Detection and Analysis of Degarelix Acetate Impurities

[0080] (1) Solution Preparation

[0081] Preparation of the test solution: Take 10 mg of the degarelix acetate sample and place it in a 100 mL volumetric flask. Add the diluent (0.1% trifluoroacetic acid solution - acetonitrile (80:20)) to dissolve and dilute to the mark, and shake well.

[0082] Impurity reference solution: Weigh approximately 5 mg each of the impurity I, impurity II, and impurity III samples and place them in a 100 mL volumetric flask and mix. Add the diluent (0.1% trifluoroacetic acid solution - acetonitrile (80:20)) to dissolve and dilute to the mark, and shake well. Accurately measure 1.0 mL and place it in a 50 mL volumetric flask, and dilute to the mark with the diluent, and shake well.

[0083] (2) Chromatographic Conditions

[0084] Chromatographic column: Chromatographic column YMC-Pack ODS AQ filled with octadecylsilyl silica gel (4.6×250mm, 5μm);

[0085] Detector: Ultraviolet detector;

[0086] Detection wavelength: 226nm;

[0087] Mobile phase A: Measure 920 ml of buffer solution (buffer solution: 0.02 mol / L sodium dihydrogen phosphate solution and 0.025 mol / L disodium hydrogen phosphate solution, pH = 2.5), add 1 mL of trifluoroacetic acid and mix well, then add 80 mL of acetonitrile;

[0088] Mobile phase B: Acetonitrile;

[0089] Flow rate: 0.9 - 1.0 mL / min;

[0090] Column temperature: 50 - 55°C;

[0091] Injection volume: 20 μL;

[0092] The gradient elution conditions are shown in Table 3.

[0093] Table 3 Gradient elution conditions

[0094] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 30 30 40 60 35 0 70 36 60 30 45 60 30

[0095] (3) Calculation

[0096] Calculation method: Use the external standard method to calculate the content of each substance in the test sample;

[0097] Calculation formula:

[0098] Concentration of impurity reference substance (mg / mL) = (weight taken (mg) × content) / dilution factor

[0099] Impurity reference substance F = (concentration of reference substance (mg / mL)) / peak area

[0100] Impurity content (%) = (average F of reference substance × peak area × dilution factor) / weight taken of sample × 100%. The content of degarelix acetate in the test sample is calculated to be 99.8%, the content of impurity I is 0.04%, the content of impurity II is not detected, and the content of impurity III is not detected.

[0101] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for detecting impurities in degarelix acetate, characterized in that: The detection method comprises the following steps: Step S1, dissolving the sample to be tested in trifluoroacetic acid-acetonitrile solution as a solvent to obtain a test solution; Step S2, dissolving the impurity standard substance with trichloroacetic acid-acetonitrile solution as solvent to obtain an impurity reference substance solution; Step S3, respectively detecting the test solution and the impurity reference solution by high performance liquid chromatography, and obtaining the type and content of impurities in the sample to be tested by comparing and analyzing the chromatograms of the test solution and the impurity reference solution, wherein the high performance liquid chromatography uses octadecylsilane bonded silica gel as filler and trifluoroacetic acid-acetonitrile system as mobile phase.

2. The detection method according to claim 1, characterized in that: In step S1 and step S2, the concentration of trifluoroacetic acid in the trifluoroacetic acid-acetonitrile solution is 0.1%, and the volume ratio of trifluoroacetic acid to acetonitrile is 80:

20.

3. The detection method according to claim 1, characterized in that: In step S1, the concentration of the sample to be tested in the test solution is 1 mg / mL.

4. The detection method according to claim 1, characterized in that: In step S1, the sequence structural formula of the impurity is one or more of the following: (1)Ac-[D-2-Nal]-[D-4-Cpa]-[D-3-Pal]-Ser-[4-Aph(D-Hor)]-[D-4-Aph(Cbm)]-Leu-[Lys(ipr)]-Pro-[D-Ala]-NH2; (2)Ac-D-2-Nal-D-4-Cpa-3-Pal-Ser-4-Aph(L-Hor)-D-4-Aph(Cbm)-Leu-Lys(ipr)-Pro-D-Ala-NH2; (3) Ac-D-2-Nal-D-4-Cpa-D-3-Pal-Ser-4-Aph(L-Hor)-D-4-Aph(Cbm)-Leu-D-Lys(ipr)-Pro-D-Ala-NH2.

5. The detection method according to claim 1, characterized in that: In step S3, the pore size of the packing material of the HPLC is 6. The detection method according to claim 1, characterized in that: In step S3, the particle size of the filler of the high performance liquid chromatography is 3-5 μm.

7. The detection method according to claim 1, characterized in that: In step S3, the filler of the high performance liquid chromatography is an end-capped filler.

8. The detection method according to claim 1, characterized in that: In step S3, the high performance liquid chromatography uses an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 226 nm.

9. The detection method according to claim 1, characterized in that: In step S3, the high performance liquid chromatography uses trifluoroacetic acid-acetonitrile solution as mobile phase A and acetonitrile as mobile phase B, and trifluoroacetic acid and acetonitrile in the mobile phase A are mixed in a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system.

10. The detection method according to claim 9, characterized in that: In step S3, the high performance liquid chromatography uses gradient elution, and the conditions of the gradient elution are as follows: 0 min, 60% by volume of mobile phase A and 30% by volume of mobile phase B; 30 min, 40% by volume of mobile phase A and 60% by volume of mobile phase B; 35 min, 70% by volume of mobile phase B; 36-45 min, 60% by volume of mobile phase A and 30% by volume of mobile phase B.

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