Method for simultaneously detecting sodium ions, ammonium ions and triethylamine in polypeptide

Abstract

The invention relates to the field of pharmaceutical analytical chemistry, and provides a method for simultaneously detecting sodium ions, ammonium ions and triethylamine in polypeptide, which is characterized by comprising the following steps: 1) respectively preparing sodium ion, ammonium ion and triethylamine reference substance solutions by using a diluent; 2) dissolving the polypeptide by using the diluent to prepare a test solution; (3) detecting the reference substance solution and the test solution by using a liquid chromatography; wherein the chromatographic conditions are as follows: a chromatographic column is an ion chromatographic column taking quaternary ammonium group bonded silica gel as a stationary phase; the detector is a non-suppression type conductivity detector; a mobile phase is a mixed solution of a nitric acid aqueous solution containing 18-crown-6-ether and acetonitrile; the elution mode is isocratic elution. According to the method, ammonium ions can be accurately detected in the presence of high-concentration sodium ions, meanwhile, the residual solvent triethylamine can be detected, detection procedures are reduced, and the method is simple in step, short in analysis period, high in specificity, high in sensitivity and good in recovery rate.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analytical chemistry, and more particularly to a method for simultaneously detecting sodium ions, ammonium ions and triethylamine in a polypeptide. Background Art

[0002] In peptide synthesis, reagents such as triethylamine, ammonia, sodium hydroxide, and ammonium bicarbonate are commonly used. According to regulations such as the United States Pharmacopoeia, the Chinese Pharmacopoeia, and the International Council for Harmonization of Pharmaceutical Codex (ICH), triethylamine is a Class III residual solvent. As a volatile amine, triethylamine is a typical low-boiling-point small molecule compound and one of the most commonly used organic bases in organic synthesis. It has potential hepatotoxicity and may cause skin, eye, and respiratory irritation. Furthermore, ammonia and ammonium bicarbonate generate ammonium ions, while sodium hydroxide generates sodium ions. Excessive intake of ammonium and sodium ions can lead to liver dysfunction and renal failure, increasing the risk of heart disease, stroke, and kidney disease. To optimize product quality, residual levels of sodium ions, ammonium ions, and triethylamine must be monitored.

[0003] While there have been reports of triethylamine being detected using gas chromatography, due to the properties of volatile amines, its detection on gas chromatography using a hydrogen flame detector is often unsatisfactory. Furthermore, there are currently no reports of simultaneous detection of sodium ions, ammonium ions, and triethylamine. Detection can only be performed on triethylamine or cations alone, which undoubtedly increases the complexity of the detection process. Furthermore, detecting ammonium ions in the presence of high sodium ion concentrations is another challenge.

[0004] In view of this, it is necessary to develop a simple and rapid method that can accurately detect sodium ions, ammonium ions and triethylamine at the same time. Summary of the Invention

[0005] To address the above technical problems, the present invention provides a method for simultaneously detecting sodium ions, ammonium ions, and triethylamine in a polypeptide. This method reduces the number of analyses and shortens the time for a single analysis to 10 minutes, significantly reducing the workload and cost of testing. Furthermore, the method enables accurate detection of ammonium ions in the presence of high sodium ion concentrations. The method comprises the following steps: 1) Prepare sodium ion, ammonium ion, and triethylamine reference solutions using diluents. 2) dissolving the polypeptide in the diluent to prepare a test solution; 3) testing the reference solution and the test solution using liquid chromatography; The chromatographic conditions include: Chromatographic column: ion chromatography column with quaternary ammonium group bonded silica gel as stationary phase; Detector: non-suppressed conductivity detector; Mobile phase: a mixture of aqueous nitric acid and acetonitrile containing 10 mM 18-crown-6-ether; Elution mode: isocratic elution.

[0006] Preferably, the diluent is ultrapure water.

[0007] Preferably, the chromatographic column is a Shodex IC YK-421 chromatographic column with a specification of 4.6 mm×125 mm and 5 μm.

[0008] Preferably, the mobile phase is a mixed solution of (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution): acetonitrile = 70-75:30-25 (v / v).

[0009] More preferably, the mobile phase is a mixed solution of (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution): acetonitrile = 75:25 (v / v).

[0010] Preferably, the chromatographic conditions further include: injection volume of 10 μL, column temperature of 40±5°C, and flow rate of 1.0±0.2 mL / min.

[0011] Preferably, the running time of step 3) is 10 minutes.

[0012] Preferably, the polypeptides include semaglutide, tilportide, teduglutide, retaglutide and etelcaltide.

[0013] Preferably, the chromatographic conditions include: Chromatographic column: Shodex IC YK-421 column, size 4.6 mm × 125 mm, 5 μm; Detector: non-suppressed conductivity detector; Mobile phase: a mixture of (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution): acetonitrile = 75:25 (v / v); Elution mode: isocratic elution; Injection volume: 10 µL; Column temperature: 40°C; Flow rate: 1.0 mL / min; Running time: 10 minutes.

[0014] Preferably, the contents of sodium ions, ammonium ions and triethylamine are calculated using an external standard method.

[0015] The beneficial effects of the present invention are: 1. The present method utilizes ion chromatography and optimizes chromatographic conditions to simultaneously detect sodium ions, ammonium ions, and triethylamine in polypeptides. This solves the problem of accurately detecting ammonium ions in the presence of high sodium ion concentrations and addresses the lack of simultaneous detection of inorganic cations and triethylamine in the prior art. This reduces the number of analyses and alleviates the burden on testers. Furthermore, the present method shortens the analysis time per analysis to 10 minutes, significantly shortening the analysis cycle. This allows for a shorter analysis time and significantly improves analytical efficiency per unit time.

[0016] 2. The present invention uses ultrapure water as a diluent, which is cheap, readily available, safe and harmless.

[0017] 3. This method has strong specificity, high sensitivity and good recovery rate, and can accurately and quickly determine the content of sodium ions, ammonium ions and residual solvent triethylamine in polypeptides. The method has been validated and the linear correlation coefficient R is 0.16μg / mL-96.0μg / mL for sodium ions. 2 is 1.000; the linear correlation coefficient R 2 The linear correlation coefficient R is 0.999, and the concentration range of triethylamine is 0.80μg / mL-12.0μg / mL. 2 The linearity was 0.999, and the spiked solution recovery rate test showed that the sodium ion content recovery rate was between 99%-103%, the ammonium ion content recovery rate was between 94%-100%, and the triethylamine content recovery rate was between 93%-96%, with excellent recovery rates. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the chromatogram of the reference solution; Figure 2 is the chromatogram of the test solution, Figure 2 B is the general Figure 2 A is the enlarged spectrum on the vertical axis; Figure 3 is the blank solution chromatogram; Figure 4 This is the chromatogram of a single-standard sodium ion solution; Figure 5 This is the chromatogram of a single standard solution of ammonium ions; Figure 6 This is the chromatogram of triethylamine single standard solution; Figure 7 is the chromatogram of the test sample spiked solution, Figure 7 B is the general Figure 7 A is the enlarged spectrum on the vertical axis; Figure 8 is the linear relationship diagram of sodium ions; Figure 9 is the linear relationship diagram of ammonium ion; Figure 10 is the linear relationship diagram of triethylamine; Figure 11 This is the chromatogram of the reference solution when the concentration of 18-crown-6 ether in the mobile phase is 5 mM; Figure 12 The chromatogram of the reference solution was detected when the mobile phase was 50% (4 mM phosphoric acid + 10 mM 18-crown-6-ether aqueous solution) + 50% acetonitrile (v / v); Figure 13 The chromatogram of the reference solution was detected when the mobile phase was 70% (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution) + 30% acetonitrile (v / v); Figure 14 The chromatogram of the reference solution was detected when the mobile phase was 50% (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution): 50% methanol (v / v). DETAILED DESCRIPTION

[0019] The present invention is further described below with reference to the accompanying drawings and examples, but these examples are exemplary and are only intended to facilitate further understanding of the features of the present invention by those skilled in the art, and do not constitute any limitation on the scope of the present invention. The reagents used in the present invention are all conventional commercially available products unless otherwise specified. The percentages appearing in the present invention are all by mass percentages unless otherwise specified. The ion chromatograph host used in the following examples is Shimadzu's LC-20A, equipped with a non-suppressed conductivity detector; the chromatographic column used is a Shodex IC YK-421 chromatographic column produced by Risenoko Co., Ltd., with a specification of 4.6 mm × 125 mm, 5 μm. In the following table, "ND" means not detected, and "N / A" means not applicable.

[0020] In the present specification, "polypeptide" refers to a polymer composed of three or more amino acids covalently linked by peptide bonds (amide bonds formed by the condensation of the carboxyl group of one amino acid with the amino group of another, followed by the removal of a water molecule). This also includes products obtained by modification during or after the synthesis of such polymers. Modifications may include the addition of side chains through substitution reactions, reaction with acids or bases to form pharmaceutically acceptable salts, or esterification reactions to form esters. The polypeptides detected using the methods of the present invention are those synthesized using triethylamine or reagents containing sodium and ammonium ions. Preferably, the polypeptides of the present invention are APIs that do not contain other excipients (e.g., surfactants, pH adjusters, stabilizers, etc.). Polypeptides suitable for use in the methods of the present invention include water-soluble polypeptides such as semaglutide, telpotide, teduglutide, retaglutide, and etelcaltide. Semaglutide is a novel, long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA). Its structure is similar to native human glucagon-like peptide-1 (GLP-1), sharing up to 94% amino acid sequence identity. It is used for glycemic control in adults with type 2 diabetes. Its primary mechanism is to activate the GLP-1 (glucagon-like peptide-1) receptor, promoting insulin secretion in a glucose-dependent manner, inhibiting glucagon secretion, and delaying gastric emptying to increase satiety. Furthermore, it suppresses appetite by inhibiting the hypothalamic feeding center, thereby achieving the effects of lowering blood sugar and reducing weight. Its amino acid sequence is as follows: H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-G1u(α-Octadecanedioic))-Glu-Phe-Ile-Ala-Trp 31-Leu-Val-Arg-Gly-Arg-Gly-OH. Tilportide is the first FDA-approved dual agonist of the glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. By activating these two receptors, it regulates blood glucose levels, increases insulin secretion, inhibits glucagon secretion, and delays gastric emptying, thereby reducing food intake and weight loss. Its primary indications are the treatment of type 2 diabetes and weight loss. In particular, its efficacy in weight loss is comparable to that of gastric resection surgery, reaching a vast market and potentially addressing the widespread obesity and suboptimal health conditions. Its amino acid sequence is as follows: Tyr-{Aib}-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{Aib}-Leu-Asp-Lys-Ile-Ala-Gln-{diacid-C20-gamma-Glu-(AEEA)2-Lys}-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2. Teduglutide is a glucagon-like peptide-2 (GLP-2) analog that reduces gastric emptying and secretion and regulates the growth, proliferation, and repair of small intestinal lining cells. It is clinically used to treat short bowel syndrome in adults. Its amino acid sequence is: His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp. Retaglutide is a triple (GLP-1R / GCGR / GIPR) agonist developed based on the GLP / GIP dual receptor agonist tielpotide by incorporating a hybrid glucagon (Gcg)-related structure. It is a drug in development for the treatment of obesity and type 2 diabetes. This active compound activates receptor agonists in the human body, contributing to blood sugar control and weight loss management. The structure of retaglutide is as follows: H-Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-γ-Glu-eicosanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2.Etecalcitide is a calcium-sensing receptor (CaSR) agonist, also known as a calcimimetics. It regulates the secretion of parathyroid hormone (PTH) via the CaSR on the surface of parathyroid chief cells. It is indicated for the treatment of post-dialysis SHPT in adults with chronic kidney disease (CKD). Its amino acid sequence is as follows: NH2-. D -Ala- D -Pbf-Arg- D -Pbf-Arg- D -Pbf-Arg- D -Ala- D -Pbf-Arg.

[0021] The present invention is described in detail below using semaglutide as an example.

[0022] Example 1: Test sample detection 1.1 Solution Preparation Diluent / blank solution: ultrapure water.

[0023] Ammonium ion stock solution (0.1 mg / mL): Accurately pipette 5.0 mL of ammonium ion standard (1000 μg / mL) (Manufacturer: Spectrum Analysis, Lot No.: 20230624) into a 50 mL plastic volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0024] Ammonium ion single standard solution: Accurately pipette 1.0 mL of ammonium ion stock solution into a 25 mL plastic volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0025] Triethylamine stock solution (0.1 mg / mL): Accurately weigh 40.15 mg of triethylamine (McLean, Lot No. C15860165) into a 20 mL plastic volumetric flask. Dilute to the mark with ultrapure water and shake well. Pipette 5.0 mL of the above solution into a 100 mL plastic volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0026] Triethylamine single standard solution: Accurately pipette 1.0 mL of triethylamine stock solution into a 25 mL plastic volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0027] Sodium ion single standard solution: Accurately pipette 0.8 mL of sodium ion standard (1000 μg / mL) (manufacturer: MacLean, batch number: C16573535) into a 25 mL plastic volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0028] Reference solution: Accurately pipette 0.8 mL of sodium ion standard (1000 μg / mL), 1.0 mL of ammonium ion stock solution (0.1 mg / mL), and 1.0 mL of triethylamine stock solution (0.1 mg / mL) into a 25 mL plastic volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0029] Test solution: Accurately weigh 20 mg of semaglutide API (Fujian Jinuohoupu Biotechnology Co., Ltd.) and place it in a 25 mL plastic volumetric flask. Dissolve and dilute to the mark with ultrapure water and shake well.

[0030] 100% test sample spiked solution: Accurately weigh 20 mg of semaglutide API (Fujian Jinuohoupu Biotechnology Co., Ltd.) and place it in a 25 mL plastic volumetric flask. Dissolve it in a small amount of ultrapure water. Then add 0.8 mL of sodium ion standard (1000 μg / mL), 1.0 mL of ammonium ion stock solution (0.1 mg / mL), and 1.0 mL of triethylamine stock solution (0.1 mg / mL). Dilute to the mark with ultrapure water and shake well.

[0031] Prepare the linear solutions according to Table 1: Table 1

[0032] Limit of quantitation (LOQ) solution: Accurately pipette 0.1 mL of sodium ion single-standard solution (32 μg / mL), 1.0 mL of ammonium ion single-standard solution (4 μg / mL), and 4.0 mL of triethylamine single-standard solution (4 μg / mL) into a 20 mL plastic volumetric flask. Dilute to the mark with diluent and shake well to prepare the LOQ solution.

[0033] Limit of detection (LOD) solution 1: Accurately pipette 1.0 mL of the quantification limit solution, add 1.0 mL of the blank solution, and shake well as the detection limit solution 1.

[0034] Limit of detection (LOD) solution 2: Accurately pipette 2.0 mL of the quantification limit solution into a 10 mL plastic volumetric flask, dilute to the mark with the blank solution, and shake well as the detection limit solution 2.

[0035] Prepare the spiked test solutions according to Table 2: Table 2

[0036] 1.2 Testing conditions The test conditions are shown in Table 3: Table 3

[0037] 1.3 Detection Inject the reference solution and the test solution separately, set the detection conditions as in Section 1.2 above, and record the chromatogram. Figure 1 is the chromatogram of the reference solution. Figure 2 is the chromatogram of the test solution. The content (residual amount) of sodium ion, ammonium ion and triethylamine in the test solution was calculated according to the external standard method. The calculation formula is as follows:

[0038] A S is the peak area of ​​sodium ion, ammonium ion or triethylamine in the test solution; A R It is the mean peak area of ​​sodium ion, ammonium ion or triethylamine in the reference solution; W R is the concentration of sodium ions, ammonium ions, or triethylamine in the reference solution (mg / mL); W S is the concentration of the test sample in the test solution (mg / mL); R is the content, %.

[0039] The test was repeated three times, and the average of the three test results was taken as the final test result. The results are shown in Table 4: Table 4

[0040] Example 2: System Applicability According to the detection conditions in Section 1.2 of Example 1, the reference solution was injected 6 times and the needle was re-injected once at the end of the sequence. The results are shown in Table 5: Table 5 As shown in Table 5, the system adaptability of the method of the present invention is good.

[0041] Example 3: Specificity According to the test conditions in Section 1.2 of Example 1, the blank solution, each single-standard solution, and 100% test sample spiked solution were injected once for testing, and the chromatograms were recorded. Figure 3-7 The chromatograms are as follows: blank solution, sodium ion single-standard solution, ammonium ion single-standard solution, triethylamine single-standard solution, and 100% test sample spiked solution. The results are summarized in Table 6: Table 6

[0042] From the above results, it can be seen that the blank solution does not affect the detection of sodium ions, ammonium ions and triethylamine. The retention times of sodium ions, ammonium ions and triethylamine in the reference solution, the test solution and the test spiked solution correspond to the retention times of sodium ions, ammonium ions and triethylamine in each single standard solution, respectively. The minimum separation of sodium ions, ammonium ions and triethylamine is 2.4, and they do not affect each other, which proves that the method of the present invention has good specificity.

[0043] Example 4: Linear According to the detection conditions in Section 1.2 of Example 1, the linear solutions were injected once each. The results are shown in Tables 7-9: Table 7

[0044] Table 8

[0045] Table 9

[0046] The standard curves were drawn with the concentrations of sodium ions, ammonium ions and triethylamine as the horizontal coordinates and the peak area as the vertical coordinates. It can be seen that the linear correlation coefficient R 2 is 1.000; the linear correlation coefficient R 2 The linear correlation coefficient R is 0.999, and the concentration range of triethylamine is 0.80μg / mL-12.0μg / mL. 2 The linear relationship is 0.999, and the linearity is good. Figure 8-10 shown.

[0047] Example 5: Detection Limit According to the detection conditions of Section 1.2 of Example 1, the detection limit solutions 1 and 2 prepared in Example 1 were injected and tested respectively. The results showed that the determination results of the detection limit solution 2 met the standards, as shown in Table 10. Table 10

[0048] Example 6: Limit of Quantitation According to the detection conditions of Section 1.2 of Example 1, the quantitative limit solution prepared in Example 1 was repeatedly injected 6 times for detection. The results are shown in Tables 11-13: Table 11

[0049] Table 12

[0050] Table 13

[0051] Example 7: Accuracy According to the preparation method of the spiked solution in Example 1, three portions of spiked solution at each concentration level were prepared, and then the test conditions in Section 1.2 of Example 1 were followed. The results are shown in Tables 14-16: Table 14

[0052] Table 15

[0053] Table 16

[0054] It can be seen from Tables 14-16 that the recovery rate of sodium ion content is between 99%-103%, the recovery rate of ammonium ion content is between 94%-100%, and the recovery rate of triethylamine content is between 93%-96%, and the RSDs are all less than 2%, indicating good recovery rates.

[0055] Example 8: Repeatability According to the method of "1.1 Preparation of Solution" in Example 1, 6 portions of 100% test sample spiked solutions were prepared. Then, according to the test conditions of Section 1.2 of Example 1, the same experimenter used the same instrument on the same day to test. The results are shown in Tables 17-19: Table 17

[0056] Table 18

[0057] Table 19

[0058] As can be seen from Tables 17-19, when 6 parallel solutions were tested, the RSDs of the test results for sodium ions, ammonium ions, and triethylamine were all less than or equal to 2.0%, indicating excellent repeatability.

[0059] Example 9: Intermediate Precision A different experimenter, on a different date than in Example 8, prepared six 100% test sample spiked solutions according to the method in "1.1 Solution Preparation" of Example 1. Testing was then performed according to the testing conditions in Section 1.2 of Example 1. The results are shown in Tables 20-22: Table 20

[0060] Table 21

[0061] Table 22

[0062] As can be seen from Tables 20-22, 6 parallel solutions were tested by different experimenters on different days, and the RSDs of the test results of sodium ions, ammonium ions and triethylamine were all less than 2.0%, and the RSDs calculated by combining the test results of the 6 solutions in Example 8 were all less than 3%. The intermediate precision of the present invention is excellent.

[0063] Example 10: Solution stability The reference solution and the 100% test sample spiked solution prepared in Example 1 were stored under analytical conditions (room temperature), sampled at different time points, and tested according to the test conditions in Section 1.2 of Example 1. The peak area change rate of each target substance (sodium ion, ammonium ion, and triethylamine) in the reference solution and the content change rate of each target substance in the 100% test sample spiked solution were calculated. The results are shown in Tables 23-24: Table 23 Peak area change rate of target peak in reference solution

[0064] Table 24 Change rate of target compound content in 100% test sample spiked solution

[0065] The rate of change of the peak area at the nth hour relative to the peak area at 0h is: (peak area tn - peak area t0) / peak area t0, where peak area tn is the peak area at the nth hour and peak area t0 is the peak area at 0h. The rate of change of the content at the nth hour relative to 0h is: (content tn - content t0) / content t0, where content tn is the content at the nth hour and content t0 is the content at 0h. As can be seen from Tables 23-24, the rate of change of the peak area of ​​each target substance in the reference solution within 30h is less than 3%, and the rate of change of the content of each target substance in the 100% test sample spiked solution within 29h is less than 2%. The solution used in the method of the present invention is stable within 30h.

[0066] Example 11: Durability The durability of the method of the present invention was verified mainly from different column temperatures. The conditions were adjusted according to Table 25 below. Except for the conditions listed in Table 25, the other conditions were consistent with those in Example 1.2.

[0067] Table 25

[0068] The experiment was conducted under the conditions listed in Table 25 above, with one injection of 100% test sample spiked solution for each condition. The results are shown in Tables 26-28: Table 26

[0069] Table 27

[0070] Table 28

[0071] It can be seen from Tables 26-28 that under different detection conditions, the content change rate of each target is less than 3%, and changes in flow rate and column temperature have no obvious effect on the test results, indicating good durability.

[0072] Comparative Example 1: The concentration of the 18-crown-6 ether aqueous solution in the mobile phase in Example 1 was adjusted to 2 mM, 4 mM, and 5 mM. The remaining conditions were the same as those in Section 1.2 of Example 1. The reference solution was tested. The results are shown in Table 29: Table 29

[0073] Figure 11 The chromatogram of the reference solution after injection is shown when the concentration of 18-crown-6 ether is 5 mM. Figure 11 As can be seen from Table 29, when the concentration of 18-crown-6-ether is lower than 10 mM, the separation between sodium ions and ammonium ions is small, which is not conducive to the detection of ammonium ions under high sodium ion concentration conditions.

[0074] Comparative Example 2 The following mobile phase was used to replace the mobile phase in Example 1. The remaining conditions were the same as those in Section 1.2 of Example 1. The reference solution was tested. The results are shown in Table 30: Table 30

[0075] The chromatogram when the mobile phase is 50% (4 mM phosphoric acid + 10 mM 18-crown-6-ether aqueous solution) + 50% acetonitrile (v / v) is as follows Figure 12 As shown in Table 30 and Figure 12It can be seen that when using phosphoric acid, triethylamine cannot elute at an acetonitrile ratio of 25%. The acetonitrile ratio must be adjusted to 50% to achieve earlier triethylamine eluent. However, at this point, not only is the triethylamine peak shape poor, but the eluent time is also prolonged, making it difficult to accurately and rapidly detect triethylamine. Furthermore, the resolution between sodium ions and ammonium ions (1.536) is also low, making it difficult to detect ammonium ions under conditions of high sodium ion concentrations.

[0076] Comparative Example 3 The proportion of acetonitrile in the mobile phase in Example 1 was adjusted to 10% (v / v), 30% (v / v), and 50% (v / v), respectively. The remaining conditions were the same as in Section 1.2 of Example 1, and the reference solution was tested. When 10% acetonitrile was used, no triethylamine peak was observed within a ten-minute run time. When 50% (v / v) acetonitrile was used, the triethylamine peak shape was poor and the peak elution time was also long. When 25% (v / v) acetonitrile and 30% (v / v) acetonitrile in Example 1 were used, the triethylamine peak shape was better and the peak elution time was faster. In particular, when 25% (v / v) acetonitrile in Example 1 was used, the triethylamine peak shape was optimal, which could minimize the analysis time, reduce analysis costs and pressure, and the separation between sodium ions and ammonium ions was also optimal. Figure 13 The chromatogram when 30% (v / v) acetonitrile was used is shown.

[0077] Comparative Example 4 The acetonitrile in the mobile phase of Example 1 was replaced with methanol, and 50% (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution): 50% methanol (v / v) was used as the mobile phase. The other conditions were the same as those in Section 1.2 of Example 1. The reference solution was tested. The results were as follows. Figure 14 As shown, it can be seen that the triethylamine peak is not only very late, but also has a poor peak shape.

[0078] It can be seen from the above examples and comparative examples that the present invention can simultaneously detect sodium ions, ammonium ions and triethylamine in polypeptides by optimizing detection conditions such as the chromatographic column and the mobile phase, and the method of the present invention has strong specificity, high sensitivity, good precision, excellent stability, strong durability and high accuracy.

[0079] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting sodium ions, ammonium ions and triethylamine in a polypeptide, characterized in that: The method comprises the following steps: 1) Prepare sodium ion, ammonium ion, and triethylamine reference solutions using diluents. 2) dissolving the polypeptide in the diluent to prepare a test solution; 3) testing the reference solution and the test solution using liquid chromatography; The chromatographic conditions include: Chromatographic column: ion chromatography column with quaternary ammonium group bonded silica gel as stationary phase; Detector: non-suppressed conductivity detector; Mobile phase: a mixture of aqueous nitric acid and acetonitrile containing 10 mM 18-crown-6-ether; Elution mode: isocratic elution.

2. The method according to claim 1, characterized in that The diluent is ultrapure water.

3. The method according to claim 1, wherein The chromatographic column is a Shodex IC YK-421 chromatographic column with a specification of 4.6 mm×125 mm and 5 μm.

4. The method according to claim 1, wherein The mobile phase is a mixed solution of (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution): acetonitrile = 70-75:30-25 (v / v).

5. The method according to claim 4, wherein The mobile phase is a mixed solution of (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution): acetonitrile = 75:25 (v / v).

6. The method according to claim 1, wherein The chromatographic conditions also include: injection volume of 10 μL, column temperature of 40±5°C, and flow rate of 1.0±0.2 mL / min.

7. The method according to claim 1, wherein The running time of step 3) is 10 min.

8. The method according to claim 1, characterized in that The polypeptides include semaglutide, tilportide, teduglutide, retaglutide and etelcaltide.

9. The method according to claim 1, wherein The chromatographic conditions include: Chromatographic column: Shodex IC YK-421 column, size 4.6 mm × 125 mm, 5 μm; Detector: non-suppressed conductivity detector; Mobile phase: a mixture of (4 mM nitric acid + 10 mM 18-crown-6-ether aqueous solution): acetonitrile = 75:25 (v / v); Elution mode: isocratic elution; Injection volume: 10 µL; Column temperature: 40°C; Flow rate: 1.0 mL / min; Running time: 10 minutes.

10. The method according to any one of claims 1 to 9, characterized in that The contents of sodium ions, ammonium ions and triethylamine were calculated using the external standard method.

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