A detection and analysis method for linaclotide and its related substances

Abstract

The present invention relates to a detection and analysis method for linaclotide and its related substances, and belongs to the field of medical technology. The present invention improves the mobile phase formula in the traditional liquid phase system, adjusts the mobile phase gradient, and improves the detection sensitivity and accuracy of the method. The method of the present invention is simple and fast, can effectively separate the known impurities of linaclotide, can accurately detect the content of each impurity, and also provides a basis for formulating the quality standard of linaclotide raw materials.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a detection and analysis method for linaclotide and related substances thereof. Background Art

[0002] Linaclotide is a guanylate cyclase C (GC-C) receptor agonist developed by Ironwood Corporation of the United States. Its trade name is Linzess. It was approved for marketing by the US FDA on August 30, 2012. Its English name is linaclotide. The drug is used to treat irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation (CIC) in adults. It is the first drug for treating constipation with this mechanism of action.

[0003] The structure of linaclotide is a polypeptide containing 14 amino acids, and its sequence is L-cysteinyl-L-cysteinyl-L-glutamyl-L-tyrosyl-L-cysteinyl-Lcysteinyl-L-asparaginyl-L-prolyl-L-alanyl-L-cysteinylL-threonyl-L-glycyl-L-cysteinyl-L-tyrosine, cyclic (1-6), (2-10), (5-13)-tris(disulfide); linaclotide is a white to off-white amorphous powder, slightly soluble in water and sodium chloride aqueous solution (0.9%). The drug exerts its effects by binding to the local GC-C receptors in the intestinal epithelium. The activated GC-C receptors induce an increase in the concentration of cGMP in the body, which can stimulate the secretion of intestinal fluid and promote gastrointestinal activity, resulting in an increase in the number of bowel movements in patients; on the other hand, it can reduce the activity of pain nerves, thereby reducing the intestinal pain of patients.

[0004] However, in the synthesis process of chemically synthesized linaclotide, it is inevitable to introduce a series of impurities, such as isomer impurities, defective peptide impurities, etc. These impurities are collectively referred to as related substances, and their presence will seriously affect the product quality of linaclotide. Since the toxicity data of these related substance impurities are unclear, there must be certain potential risks for patients to take the medicine. Therefore, in order to ensure the quality of linaclotide products and improve the safety of patients' medication, strict quality control of linaclotide related substances has very important practical significance. And so far, no method for determining linaclotide related substances has been reported in the literature.

[0005] High performance liquid chromatography (HPLC) is an efficient and rapid analytical separation technology that developed rapidly in the 1970s. It is an important means of modern separation testing and is currently the most widely used chromatographic analysis method. The high performance liquid chromatography system consists of a mobile phase liquid storage bottle, an infusion pump, an injector, a chromatographic column, a detector and a recorder.

[0006] The separation principle of HPLC is: when the components dissolved in the mobile phase pass through the stationary phase, they have different retention times in the stationary phase due to the different sizes and strengths of their interactions (adsorption, distribution, exclusion, affinity) with the stationary phase, and thus flow out of the stationary phase one after another. In actual operation, the retention value and selectivity of the sample in the chromatographic column are adjusted mainly by changing the composition of the mobile phase, so that different samples can be separated.

[0007] However, there are no reports on the detection of linaclotide and its related substances using HPLC. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides a method for detecting linaclotide and its related substances. The detection and analysis method is a method for detecting impurities (impurity A, impurity B, impurity C, impurity D and impurity E) in linaclotide raw materials. The mobile phase formula in the traditional liquid phase system is improved, the mobile phase gradient is adjusted, and the detection sensitivity and accuracy of the method are improved.

[0009] Among them, impurity A is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-OHcyclic(1-6),(2-10),(5-13)-tri(disulfide).

[0010] Impurity B is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asp-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide).

[0011] Impurity C is Ac-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide).

[0012] Impurity D is H-Cys-Cys-Glu-Tyr-D-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide).

[0013] Impurity E is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Ala-Cys-Thr-Gly-Cys-Tyr-OH cyclic(1-6),(2-11),(5-14)-tris(disulfide).

[0014] The present invention is achieved through the following technical solutions:

[0015] The purpose of the present invention is to provide a method for detecting and analyzing linaclotide and its related substances, comprising the following steps:

[0016] (1) Preparation of localization solution: diluting linaclotide and impurities with diluents to prepare localization solution;

[0017] (2) Preparation of sample solution: diluting the sample to be tested with a diluent to prepare a solution to be tested;

[0018] (3) High performance liquid chromatography analysis: performing high performance liquid chromatography on the positioning solution obtained in step (1), the linaclotide solution obtained in step (2), and the impurity solution, and then performing quantitative and / or qualitative analysis;

[0019] The high performance liquid chromatography comprises the following conditions:

[0020] Mobile phase A is a mixed solution of sodium perchlorate and potassium dihydrogen phosphate, wherein the molar ratio of sodium perchlorate to potassium dihydrogen phosphate is 0.5-1.5:1;

[0021] Mobile phase B was methanol;

[0022] The mobile phase gradient elution program was: 0 min, 5% B; 0 min-10 min, 5%-10% B; 10 min-43 min, 10%-38% B; 43 min-55 min, 38%-80% B; 55 min-56 min, 80%-5% B; 56 min-65 min, 5% B;

[0023] The chromatographic column is a Phenomenex kinetexps C18 column, the particle size of the chromatographic column is 2.6 μm, the specification is 150 mm×4.6 mm, the column temperature is 30° C.-40° C., the wavelength is 220 nm, and the flow rate is 0.5 mL / min-0.8 mL / min.

[0024] In one embodiment of the present invention, the diluent in step (1) and step (2) is a mixture of acetonitrile and hydrochloric acid solution.

[0025] In one embodiment of the present invention, the volume ratio of acetonitrile to hydrochloric acid solution is 1-3:3-5.

[0026] In one embodiment of the present invention, the volume ratio of acetonitrile to hydrochloric acid solution is 1-2:3-4.

[0027] In one embodiment of the present invention, the volume ratio of acetonitrile to hydrochloric acid solution is 3:7.

[0028] In one embodiment of the present invention, the concentration of the hydrochloric acid solution is 0.05 mol / L-0.2 mol / L.

[0029] In one embodiment of the present invention, in step (1), the impurities are impurity A, impurity B, impurity C, impurity D and impurity E;

[0030] Wherein, impurity A is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide);

[0031] Impurity B is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asp-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide);

[0032] Impurity C is Ac-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide);

[0033] Impurity D is H-Cys-Cys-Glu-Tyr-D-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide);

[0034] Impurity E is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Ala-Cys-Thr-Gly-Cys-Tyr-OH cyclic(1-6),(2-11),(5-14)-tris(disulfide).

[0035] In one embodiment of the present invention, in step (2), the concentration of linaclotide in the test solution is 0.1 mg / mL-0.3 mg / mL.

[0036] In one embodiment of the present invention, in step (2), the concentration of impurities in the test solution is 0.1 μg / mL-0.3 μg / mL.

[0037] In one embodiment of the present invention, the pH value of the mobile phase A is 4.0-5.5.

[0038] In one embodiment of the present invention, the pH value of the mobile phase A is 4.5-5.5.

[0039] In one embodiment of the present invention, the pH value of the mobile phase A is 5.0.

[0040] In one embodiment of the present invention, the detection and analysis method comprises the following steps:

[0041] (1) Preparation of test solution: dilute linaclotide with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution at a volume ratio of 3:7 to prepare a solution containing 0.2 mg per 1 mL, which is the test solution;

[0042] (2) Take 50 μL of the test sample solution, analyze it by liquid chromatography, record the chromatogram, and calculate the related substances of the test sample by the area normalization method.

[0043] Chromatographic conditions:

[0044] Chromatographic column: Phenomenex kinetexps C18 column (150mm×4.6mm, 2.6μm), 0.05mol / L sodium perchlorate and 0.05mol / L sodium acetate solution (pH5.0) as mobile phase A, methanol as mobile phase B, detection wavelength 220nm; flow rate 0.6mL / min, gradient elution.

[0045] Detection method of related substances: The chromatographic conditions of the high performance liquid chromatography method are as follows: a chromatographic column with octadecylsilane bonded silica gel as the filler, mobile phase A is 0.05 mol / L sodium perchlorate and 0.05 mol / L potassium dihydrogen phosphate solution (pH 4.0-6.0); mobile phase B is methanol; flow rate is 0.5 mL / min-0.8 mL / min; column temperature is 30°C-40°C, wavelength is 220 nm, and gradient elution is performed.

[0046] The present invention uses a new special stationary phase filler chromatographic column, the chromatographic column filler includes a core-shell filler, and the filler surface is positively charged. When using the traditional hydrocarbon C18 stationary phase to analyze alkaline substances, tailing peaks often appear due to secondary interactions on the silica gel surface. The Kinetex PS C18 surface has a positive charge, which can effectively repel strong bases, making the peak shape better and more reproducible. This type of chromatographic column has higher selectivity and sensitivity, and under specific chromatographic conditions, it can more accurately and effectively determine the content of related substances in linaclotide.

[0047] The technical solution of the present invention has the following advantages:

[0048] (1) The present invention discloses a method for detecting and analyzing linaclotide and its related substances, which improves the detection sensitivity and accuracy of the method by improving the mobile phase formula in the traditional liquid phase system and adjusting the mobile phase gradient.

[0049] (2) The detection and analysis method of linaclotide and its related substances of the present invention is simple and rapid, which can not only effectively separate the known impurities of linaclotide, but also accurately detect the content of each impurity, and also provide a basis for formulating the quality standard of linaclotide raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0051] Figure 1 It is the detection chromatogram of the diluent in Example 2 of the present invention;

[0052] Figure 2 The system suitability chromatogram in Example 3 of the present invention - mobile phase A (pH 4.0);

[0053] Figure 3 It is a partial enlarged view of the system suitability chromatogram in Example 3 of the present invention - mobile phase A (pH 4.0);

[0054] Figure 4 The system suitability chromatogram in Example 4 of the present invention - mobile phase A (pH 4.5);

[0055] Figure 5 It is a partial enlarged view of the system suitability chromatogram in Example 4 of the present invention - mobile phase A (pH 4.5);

[0056] Figure 6 The system suitability chromatogram in Example 5 of the present invention - mobile phase A (pH 5.0);

[0057] Figure 7It is a partial enlarged view of the system suitability chromatogram in Example 5 of the present invention - mobile phase A (pH 5.0);

[0058] Figure 8 The system suitability chromatogram in Example 6 of the present invention - mobile phase A (pH 5.5);

[0059] Fig. 9 It is a partial enlarged view of the system suitability chromatogram in Example 6 of the present invention - mobile phase A (pH 5.5);

[0060] Fig.10 is the system suitability chromatogram in Example 7 of the present invention;

[0061] Fig.11 It is a partial enlarged view of the system suitability chromatogram in Example 7 of the present invention;

[0062] Fig.12 This is the localization map of linaclotide in Example 7 of the present invention;

[0063] Fig.13 This is a partial enlarged view of the localization map of linaclotide in Example 7 of the present invention;

[0064] Fig.14 This is a localization diagram of impurity A in Example 7 of the present invention;

[0065] Fig.15 It is a partial enlarged view of the impurity A location map in Example 7 of the present invention;

[0066] Fig.16 This is a localization diagram of impurity B in Example 7 of the present invention;

[0067] Fig.17 It is a partial enlarged view of the impurity B localization map in Example 7 of the present invention;

[0068] Fig.18 This is the location map of impurity C in Example 7 of the present invention;

[0069] Fig.19 It is a partial enlarged view of the impurity C localization map in Example 7 of the present invention;

[0070] Fig. 20 This is a localization diagram of impurity D in Example 7 of the present invention;

[0071] Fig.21 It is a partial enlarged view of the impurity D localization map in Example 7 of the present invention;

[0072] Fig. 22 This is a localization diagram of impurity E in Example 7 of the present invention;

[0073] Fig.23 It is a partial enlarged view of the impurity E localization map in Example 7 of the present invention;

[0074] Fig.24 The chromatograms of different mobile phases in Comparative Example 1 of the present invention are shown below;

[0075] Fig.25 It is a partial enlarged view of the chromatogram of different mobile phases in Comparative Example 1 of the present invention;

[0076] Fig.26 This is a chromatogram detected using a Welch Ultimate C18 chromatographic column in Comparative Example 2 of the present invention;

[0077] Fig. 27 It is a partial enlarged view of the chromatogram detected using the Welch Ultimate C18 chromatographic column in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0078] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0079] Example 1

[0080] Since different impurities have different polarities, solvents with different polarities can dissolve different impurities. Therefore, in order to ensure that the impurities in the sample are completely dissolved, the present invention studies the types of diluents.

[0081] Linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E were diluted with 0.1 mol / L hydrochloric acid solution to prepare 0.2 mg / mL linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E solutions. It was found through experiments that linaclotide, impurity A, impurity B and impurity C were completely dissolved, while impurity D and impurity E were not completely dissolved. It can be concluded that 0.1 mol / L hydrochloric acid solution is not suitable for dissolving impurities in the present invention.

[0082] Example 2

[0083] A solution obtained by mixing acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 was used as a diluent, and the diluent was subjected to chromatographic detection. The instrument and chromatographic detection conditions were as follows: Agilent 1260 Infinity II high performance liquid chromatograph, Phenomenex kinetex ps C18 column (150 mm×4.6 mm, 2.6 μm), 0.05 mol / L sodium perchlorate and 0.05 mol / L sodium acetate solution as mobile phase A, methanol as mobile phase B, gradient elution, detection wavelength of 220 nm; flow rate of 0.6 mL / min, injection volume of 50 μL; specific gradient elution: 0 min, 5% B; 0-10 min, 5%-10% B; 10-43 min, 10%-38% B; 43-55 min, 38%-80% B; 55-56 min, 80%-5% B; 56-65 min, 5% B.

[0084] The detection chromatogram of the dilution is as follows Figure 1 As shown. Figure 1 The results showed that linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E solutions were completely dissolved in the diluent. It can be concluded that a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 can be used as a detection diluent.

[0085] Example 3

[0086] Experimental instruments and chromatographic conditions: Agilent 1260 InfinityⅡ HPLC, Phenomenexkinetex ps C18 column (150 mm×4.6 mm, 2.6 μm), 0.05 mol / L sodium perchlorate and 0.05 mol / L sodium acetate solution as mobile phase A, acetic acid was used to adjust the pH of mobile phase A to 4.0, methanol was used as mobile phase B, gradient elution was performed, and the detection wavelength was 220 nm; flow rate was 0.6 mL / min, injection volume was 50 μL; the specific gradient elution program is shown in Table 1.

[0087] Table 1 Gradient elution program

[0088]

[0089] Sample preparation: Take linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E, dilute them with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 as a diluent, and prepare a solution containing 0.2 mg of linaclotide and 0.2 μg of each impurity per 1 mL, which is the system suitability solution.

[0090] The system suitability solution was tested using a high performance liquid chromatograph, and the detection chromatogram of mobile phase A with a pH of 4.0 was as follows: Figure 2 and Figure 3 As shown, Figure 3 for Figure 2 A partial enlargement of the chromatogram.

[0091] Example 4

[0092] Experimental instruments and chromatographic conditions: Agilent 1260 InfinityⅡ HPLC, Phenomenexkinetex ps C18 column (150 mm×4.6 mm, 2.6 μm), 0.05 mol / L sodium perchlorate and 0.05 mol / L sodium acetate solution as mobile phase A, acetic acid was used to adjust the pH of mobile phase A to 4.5, methanol was used as mobile phase B, gradient elution was performed, and the detection wavelength was 220 nm; flow rate was 0.6 mL / min, injection volume was 50 μL; the specific gradient elution program is shown in Table 1.

[0093] Sample preparation: Take linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E, dilute them with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 as a diluent, and prepare a solution containing 0.2 mg of linaclotide and 0.2 μg of each impurity per 1 mL, which is the system suitability solution.

[0094] The system suitability solution was tested using a high performance liquid chromatograph, and the detection chromatogram of mobile phase A with a pH of 4.5 was as follows: Figure 4 and Figure 5 As shown, Figure 5 for Figure 4 A partial enlargement of the chromatogram.

[0095] Example 5

[0096] Experimental instruments and chromatographic conditions: Agilent 1260 InfinityⅡ HPLC, Phenomenexkinetex ps C18 column (150 mm×4.6 mm, 2.6 μm), 0.05 mol / L sodium perchlorate and 0.05 mol / L sodium acetate solution as mobile phase A, acetic acid was used to adjust the pH of mobile phase A to 5.0, methanol was used as mobile phase B, gradient elution was performed, and the detection wavelength was 220 nm; flow rate was 0.6 mL / min, injection volume was 50 μL; the specific gradient elution program is shown in Table 1.

[0097] Sample preparation: Take linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E, dilute them with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 as a diluent, and prepare a solution containing 0.2 mg of linaclotide and 0.2 μg of each impurity per 1 mL, which is the system suitability solution.

[0098] The system suitability solution was tested using a high performance liquid chromatograph, and the detection chromatogram of mobile phase A with a pH of 5.0 was as follows: Figure 6 and Figure 7 As shown, Figure 7 for Figure 6 A partial enlargement of the chromatogram.

[0099] Example 6

[0100] Experimental instruments and chromatographic conditions: Agilent 1260 InfinityⅡ HPLC, Phenomenexkinetex ps C18 column (150 mm×4.6 mm, 2.6 μm), 0.05 mol / L sodium perchlorate and 0.05 mol / L sodium acetate solution as mobile phase A, acetic acid was used to adjust the pH of mobile phase A to 5.5, methanol was used as mobile phase B, gradient elution was performed, and the detection wavelength was 220 nm; flow rate was 0.6 mL / min, injection volume was 50 μL; the specific gradient elution program is shown in Table 1.

[0101] Sample preparation: Take linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E, dilute them with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 as a diluent, and prepare a solution containing 0.2 mg of linaclotide and 0.2 μg of each impurity per 1 mL, which is the system suitability solution.

[0102] The system suitability solution was tested using a high performance liquid chromatograph, and the chromatogram obtained for the mobile phase A with a pH of 5.5 was as follows: Figure 8 and Fig. 9 As shown, Fig. 9 for Figure 8 A partial enlargement of the chromatogram.

[0103] According to the chromatograms of mobile phase A at different pH values ​​(pH 4.0, pH 4.5, pH 5.0 and pH 5.5) in Examples 3-6 (specifically as Figure 2-Figure 9 The specific data of the test are shown in Table 2.

[0104] Table 2 Separation of impurities in mobile phase A at different pH

[0105]

[0106] It can be seen from Table 2 that when the pH of mobile phase A is low (pH 4.0 and pH 4.5), the main peak and impurity D cannot be effectively separated; when the pH value of mobile phase A is 5.0 and 5.5, each known impurity can be effectively separated from the main peak, but it can be seen from the main peak tailing factor that the main peak has better symmetry when the pH value is 5.0. By comparison, it is found that the impurity separation is better when the pH value of mobile phase A is 5.0, so the optimal pH value of mobile phase A is determined to be 5.0.

[0107] Example 7: Impurity Location Study

[0108] Purpose of the experiment: To determine the retention time of linaclotide and each known impurity.

[0109] Experimental instruments and chromatographic conditions: Agilent 1260 Infinity II high performance liquid chromatograph, Phenomenex kinetex ps C18 column (150 mm*4.6 mm, 2.6 μm), 0.05 mol / L sodium perchlorate and 0.05 mol / L sodium acetate solution (pH 5.0) as mobile phase A, methanol as mobile phase B, gradient elution, detection wavelength 220 nm; flow rate 0.6 mL / min, injection volume 50 μL. The gradient elution program is shown in Table 1.

[0110] Sample preparation: Take linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E, dilute them with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 as a diluent, and prepare a solution containing 0.2 mg of linaclotide and 0.2 μg of each impurity per 1 mL, which is the system suitability solution.

[0111] Linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E were diluted with diluent (a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7) to prepare solutions containing 0.2 mg of linaclotide and each related substance per 1 mL, which were used as the positioning solutions.

[0112] Linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E were diluted with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 as a diluent to prepare a solution containing 0.2 mg of linaclotide and 0.2 μg of each impurity per 1 mL, which was the system suitability solution.

[0113] The system suitability solution and each positioning solution were tested according to the above chromatographic conditions. The results of the impurity positioning study are shown in Table 3 and the positioning diagram is shown in Figure 10-Figure 23 shown.

[0114] Table 3 Results of impurity location study

[0115]

[0116]

[0117] Depend on Figure 10-Figure 23 As can be seen from Table 3, the detection and analysis method of linaclotide and its related substances of the present invention can completely separate linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E by improving the diluent and the mobile phase formula in the traditional liquid phase system, and can accurately detect each known impurity, thereby improving the detection sensitivity and accuracy of the method, and providing an effective method for linaclotide quality control.

[0118] Comparative Example 1 (Replacement of mobile phase)

[0119] Experimental instruments and chromatographic conditions: Agilent 1260 InfinityⅡ HPLC, Phenomenexkinetex ps C18 column (150 mm×4.6 mm, 2.6 μm), water-acetonitrile-trifluoroacetic acid (98:2:0.1) as mobile phase A, acetonitrile-water-trifluoroacetic acid (95:5:0.1) as mobile phase B, gradient elution, detection wavelength 220 nm; flow rate 0.6 mL / min, injection volume 50 μL; the specific gradient elution program is shown in Table 1.

[0120] Sample preparation: Take linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E, dilute them with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 as a diluent, and prepare a solution containing 0.2 mg of linaclotide and 0.2 μg of each impurity per 1 mL, which is the system suitability solution.

[0121] The system suitability solution was tested using a high performance liquid chromatograph. The chromatogram was as follows: Fig.24 and Fig.25 As shown, Fig.25 for Fig.24 A partial enlargement of the chromatogram.

[0122] Comparative Example 2 (Replacing the chromatographic column)

[0123] Experimental instruments and chromatographic conditions: Agilent 1260 InfinityⅡ high performance liquid chromatograph, Welch Ultimate C18 column (150×4.6 mm, 2.6 μm), water-acetonitrile-trifluoroacetic acid (98:2:0.1) as mobile phase A, acetonitrile-water-trifluoroacetic acid (95:5:0.1) as mobile phase B, gradient elution, detection wavelength 220 nm; flow rate 0.6 mL / min, injection volume 50 μL; the specific gradient elution process is shown in Table 1.

[0124] Sample preparation: Take linaclotide, impurity A, impurity B, impurity C, impurity D and impurity E, dilute them with a mixture of acetonitrile and 0.1 mol / L hydrochloric acid solution in a volume ratio of 3:7 as a diluent, and prepare a solution containing 0.2 mg of linaclotide and 0.2 μg of each impurity per 1 mL, which is the system suitability solution.

[0125] The system suitability solution was tested using a high performance liquid chromatograph. The chromatogram was as follows: Fig.26 and Fig. 27 As shown, Fig. 27 for Fig.26 A partial enlarged view of the chromatogram. Fig.26 and Fig. 27 It can be seen that when the common C18 column Welch Ultimate C18 is used as the chromatographic column (150 mm×4.6 mm, 2.6 μm) and other conditions are the same as those in Example 2, impurities C and D cannot be effectively separated.

[0126] Obviously, the above embodiments are only examples for clear explanation and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the invention.

Claims

1. A method for detecting and analyzing linaclotide and its related substances, characterized in that: The following steps are involved: (1) Preparation of localization solution: dilute linaclotide and impurities with diluents to prepare localization solution; (2) Preparation of sample solution: diluting the sample to be tested with a diluent to prepare a solution to be tested; (3) High performance liquid chromatography analysis: performing high performance liquid chromatography on the positioning solution obtained in step (1), the linaclotide solution obtained in step (2), and the impurity solution, and then performing quantitative and / or qualitative analysis; The high performance liquid chromatography comprises the following conditions: Mobile phase A is a mixed solution of sodium perchlorate and potassium dihydrogen phosphate, wherein the molar ratio of sodium perchlorate to potassium dihydrogen phosphate is 0.5-1.5:1; Mobile phase B was methanol; The mobile phase gradient elution program was: 0 min, 5% B; 0 min-10 min, 5%-10% B; 10 min-43 min, 10%-38% B; 43 min-55 min, 38%-80% B; 55 min-56 min, 80%-5% B; 56 min-65 min, 5% B; The chromatographic column is a Phenomenex kinetex ps C18 column, the particle size of the chromatographic column is 2.6 μm, the specification is 150 mm×4.6 mm, the column temperature is 30° C.-40° C., the wavelength is 220 nm, and the flow rate is 0.5 mL / min-0.8 mL / min; In step (1), the impurities are impurity A, impurity B, impurity C, impurity D and impurity E; Wherein, impurity A is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-OHcyclic (1-6), (2-10), (5-13)-tris(disulfide); Impurity B is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asp-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide); Impurity C is Ac-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide); Impurity D is H-Cys-Cys-Glu-Tyr-D-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-10),(5-13)-tris(disulfide); Impurity E is H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Ala-Cys-Thr-Gly-Cys-Tyr-OHcyclic(1-6),(2-11),(5-14)-tris(disulfide); The pH value of the mobile phase A is 5.0-5.

5.

2. The detection and analysis method according to claim 1, characterized in that: The diluent in step (1) and step (2) is a mixture of acetonitrile and hydrochloric acid solution.

3. The detection and analysis method according to claim 2, characterized in that: The volume ratio of the acetonitrile to the hydrochloric acid solution is 1-5:5-9.

4. The detection and analysis method according to claim 3, characterized in that: The volume ratio of the acetonitrile to the hydrochloric acid solution is 1-2:3-4.

5. The detection and analysis method according to claim 3, characterized in that: The concentration of the hydrochloric acid solution is 0.05 mol / L-0.2 mol / L.

6. The detection and analysis method according to claim 1, characterized in that: In step (2), the concentration of linaclotide in the test solution is 0.1 mg / mL-0.3 mg / mL.

7. The detection and analysis method according to claim 1, characterized in that: In step (2), the concentration of impurities in the test solution is 0.1 μg / mL-0.3 μg / mL.

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