Stabilizer and application thereof, analysis method of easily ionized compound and regeneration method of high performance liquid chromatographic column

Abstract

The invention provides a stabilizer and application thereof, an analysis method of an easily ionized compound and a regeneration method of a high performance liquid chromatographic column, and relates to the technical field of analysis and detection. Methoxypolyethylene glycol has a certain effect of a surfactant, can cooperate with an ion pair reagent in a certain pH value range, and can inhibit ionization of an easily ionized compound (a target compound), so that the target compound exists in a neutral molecular form in a mobile phase, the purpose of separating the easily ionized compound is achieved, and the separation efficiency of the target compound is improved. The damage of the ion pair reagent to the chromatographic column is also avoided, so that the service life of the chromatographic column is prolonged. For a chromatographic column which cannot meet the detection and separation requirements of the easily ionized compounds, the chromatographic column can meet the chromatographic peak separation requirements again, and the method plays a crucial role in detection and separation of the easily ionized compounds. Moreover, the stabilizer is added into the mobile phase for high performance liquid chromatography, so that the method is simple and convenient, and the input cost of the chromatographic column can be greatly saved.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a stabilizer and application thereof, an analysis method for easily ionizable compounds and a regeneration method for a high performance liquid chromatography column. Background Art

[0002] Ionic compounds, especially easily ionized ionic compounds, need to be separated by chromatographic conditions, and ion pair reagents are needed in the mobile phase. The chromatographic column stationary phase is charged, and the principle of opposite charge attraction is used to adsorb and retain easily ionized compounds such as vecuronium bromide, rocuronium bromide, and cetylpyridinium chloride. For example, the mobile phases in the 2020 edition of the Chinese Pharmacopoeia, Part II, USPNF 2023, and EP11.4 all use ion pair reagents.

[0003] However, the above method has two obvious disadvantages: First, the ion pair reagent is adsorbed on the active sites of the chromatographic column packing, which is difficult to be washed off, thereby affecting the active sites of the stationary phase, resulting in an early retention time, a decrease in the number of theoretical plates, and a deterioration in separation. Since the adsorption process is irreversible, even if the chromatographic column is fully washed, the performance of the chromatographic column can only be partially restored, and the service life of the chromatographic column is very short, and the economic cost is relatively high. Second, the intra-batch and inter-batch differences of the chromatographic column lead to fluctuations in separation. The silica gel surface of the chromatographic column will have some chemical bonds (such as hydroxyl groups) remaining when it leaves the factory. The slight differences in the residues between batches of the chromatographic column do not have a significant impact on the separation of ordinary compounds, but for chromatographic systems with ion pair reagents, a part of the ion pair reagents are filled into the gaps of the stationary phase and bonded to it, so it is easier to show the impact of the batch differences of the chromatographic column on the separation.

[0004] Therefore, if a stabilizer can be added to improve the situation where the polarity of the chromatographic column is affected when using ion pair reagents, so that the retention time returns to normal, the number of theoretical plates increases, and the separation degree meets the requirements, it will play a vital role in the analysis of ionic compounds. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a stabilizer and its application, an analysis method for easily ionized compounds and a regeneration method for a high performance liquid chromatography column. The present invention utilizes polyethylene glycol monomethyl ether to improve the situation where the column efficiency of the chromatographic column is reduced and impurities cannot be separated.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a stabilizer, comprising polyethylene glycol monomethyl ether and a pH value regulator.

[0008] The present invention also provides the use of the stabilizer described in the above technical solution in liquid chromatography analysis of easily ionizable compounds; the analysis is detection or separation.

[0009] Preferably, the stabilizer also includes a pH adjuster.

[0010] Preferably, the easily ionizable compound comprises one or more of vecuronium bromide, rocuronium bromide, pipecuronium bromide and cetylpyridinium chloride.

[0011] The present invention also provides an analysis method for easily ionizable compounds, comprising the following steps: analyzing the easily ionizable compounds to be tested to obtain analysis results of the easily ionizable compounds; the analysis is detection or separation; the mobile phase used in the high performance liquid chromatography analysis contains the stabilizer described in the above technical solution.

[0012] Preferably, the concentration of polyethylene glycol monomethyl ether in the mobile phase is 4-6 g / L.

[0013] Preferably, the conditions for the HPLC analysis include: the filler of the chromatographic column includes octadecylsilane bonded silica gel, silica gel or cyano bonded silica gel, the detection wavelength is 210-260 nm, the mobile phase flow rate is 1-2 mL / min, the column temperature is 30-45°C, and the injection volume is 20-100 μL.

[0014] Preferably, when the easily ionizable compound is vecuronium bromide, the mobile phase of the high performance liquid chromatography analysis is a mixed solvent of a first weakly alkaline tetramethylammonium hydroxide aqueous solution, methanol, acetonitrile and polyethylene glycol monomethyl ether; the volume ratio of the first weakly alkaline tetramethylammonium hydroxide aqueous solution, methanol and acetonitrile is 120-150:235-265:600-630; the pH value of the first weakly alkaline tetramethylammonium hydroxide aqueous solution is 6.5-8.

[0015] Preferably, when the easily ionizable compound is rocuronium bromide, the mobile phase of the high performance liquid chromatography analysis is a mixed solvent of a second weak alkaline tetramethylammonium hydroxide solution, acetonitrile and polyethylene glycol monomethyl ether; the volume ratio of the second weak alkaline tetramethylammonium hydroxide solution to acetonitrile is 5-15:85-95; the pH value of the second weak alkaline tetramethylammonium hydroxide solution is 6.5-8.

[0016] Preferably, when the easily ionizable compound is cetylpyridinium chloride, the mobile phase of the high performance liquid chromatography analysis includes mobile phase A and mobile phase B; the mobile phase A is methanol; the mobile phase B is a mixed solution of tetramethylammonium hydroxide aqueous solution, potassium dihydrogen phosphate aqueous solution and polyethylene glycol monomethyl ether; the pH value of the mobile phase B is 3-4.5; the elution method is gradient elution, and the gradient elution program is as follows: 0-8 min, the volume fraction of the mobile phase A increases from 50% to 70%; 8-30 min, the volume fraction of the mobile phase A is 70%; 30-32 min, the volume fraction of the mobile phase A decreases from 70% to 50%; 32-40 min, the volume fraction of the mobile phase A is 50%.

[0017] The present invention also provides a method for regenerating a high performance liquid chromatography column, comprising the following steps: utilizing a regenerated mobile phase to regenerate the chromatography column used in the analytical method described in the above technical solution; the regenerated mobile phase contains polyethylene glycol monomethyl ether.

[0018] When using ion pair reagents, the way to adjust the retention time is different from that of ordinary reverse phase. Changing the mobile phase pH value can inhibit the ionization of easily ionized compounds (target compounds), so that the target compound exists in a neutral molecular form in the mobile phase, making the detection and separation of easily ionized compounds more effective. Polyethylene glycol monomethyl ether has a certain surfactant effect, which can synergize with ion pair reagents within a certain pH range, not only achieving the purpose of separating easily ionized compounds, but also avoiding the damage of ion pair reagents to the chromatographic column, thereby extending the life of the chromatographic column. For the chromatographic column that can no longer meet the requirements for the detection and separation of easily ionized compounds, it is made to meet the requirements for the separation of chromatographic peaks again, which plays a vital role in the detection and separation of easily ionized compounds. Moreover, the present invention is carried out by adding a stabilizer to the mobile phase for high performance liquid chromatography analysis, and the method is simple, which can greatly save the investment cost of the chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the HPLC chromatogram of (1.1) in Example 1;

[0020] Figure 2 is the HPLC chromatogram of (1.2) in Example 1;

[0021] Figure 3 is the HPLC chromatogram of (1.3) in Example 1;

[0022] Figure 4 is the HPLC chromatogram of (1.4) in Example 1;

[0023] Figure 5 is the HPLC chromatogram of (1.5) in Example 1;

[0024] Figure 6 is the HPLC chromatogram of (1.6) in Example 1;

[0025] Figure 7 is the HPLC chromatogram of (2.1) in Example 2;

[0026] Figure 8 is the HPLC chromatogram of (2.2) in Example 2;

[0027] Fig. 9 It is the high performance liquid chromatogram of (2.3) in Example 2. DETAILED DESCRIPTION

[0028] The invention provides a stabilizer, comprising polyethylene glycol monomethyl ether and a pH value regulator.

[0029] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0030] In the present invention, the pH adjuster preferably includes an acid and / or an aqueous sodium hydroxide solution; the acid preferably includes phosphoric acid and / or glacial acetic acid; the concentration of the aqueous sodium hydroxide solution is preferably 1 to 4 mol / L, more preferably 1.5 to 3.5 mol / L, and further preferably 2 to 3 mol / L.

[0031] The present invention also provides the above technical solution. The present invention provides the use of a stabilizer in liquid chromatography analysis of easily ionizable compounds; the stabilizer includes polyethylene glycol monomethyl ether; and the analysis is detection or separation.

[0032] In the present invention, the number average molecular weight of the polyethylene glycol monomethyl ether is preferably 400-1000, more preferably 500-800.

[0033] In the present invention, the easily ionizable compound preferably includes one or more of vecuronium bromide, rocuronium bromide, pipecuronium bromide and cetylpyridinium chloride.

[0034] When using ion pair reagents, the way to adjust the retention time is different from that of ordinary reverse phase. Changing the pH value is different from changing the organic phase ratio. Changing the pH value of the mobile phase can inhibit the ionization of easily ionized compounds (target compounds), so that the target compounds exist in the mobile phase in a neutral molecular form, making the detection and separation of easily ionized compounds more effective. Polyethylene glycol monomethyl ether has a certain surfactant effect and can synergize with ion pair reagents within a certain pH range, which not only achieves the purpose of separating easily ionized compounds, but also avoids damage to the chromatographic column by the ion pair reagent, thereby extending the life of the chromatographic column. For chromatographic columns that can no longer meet the detection and separation requirements of easily ionized compounds, it can make them meet the chromatographic peak separation requirements again, which plays a vital role in the detection and separation of easily ionized compounds.

[0035] In the present invention, the mobile phase for liquid chromatography detection contains polyethylene glycol monomethyl ether.

[0036] The present invention provides a method for detecting an easily ionizable compound, comprising the following steps: subjecting the easily ionizable compound to be detected to high performance liquid chromatography analysis to obtain a detection result of the easily ionizable compound; the mobile phase used in the high performance liquid chromatography analysis contains the stabilizer described in the above technical solution; and the analysis is detection or separation.

[0037] In the present invention, the concentration of polyethylene glycol monomethyl ether in the mobile phase is preferably 4 to 6 g / L, more preferably 4.5 to 5.5 g / L, and further preferably 5 g / L.

[0038] In the present invention, the easily ionizable compound includes one or more of vecuronium, rocuronium, pipecuronium and cetylpyridinium chloride. In the present invention, the vecuronium preferably includes vecuronium for injection; the rocuronium preferably includes rocuronium injection; the pipecuronium preferably includes pipecuronium for injection; the cetylpyridinium chloride preferably includes cetylpyridinium chloride gargle.

[0039] In the present invention, the conditions for the HPLC analysis include: the filler of the chromatographic column includes octadecylsilane bonded silica gel, silica gel or cyano bonded silica gel; the chromatographic column more preferably includes Thermo scientific ODSHyoersil chromatographic column, HALO Hilic column or Ultimate XB-CN column; the column is preferably equilibrated with a mobile phase for 4 to 8 hours, more preferably 4 to 6 hours, before use; the detection wavelength is 210 to 260 nm, more preferably 210 nm or 259 nm, the detection wavelength of vecuronium, rocuronium and pipecuronium is preferably 210 nm; the detection wavelength of cetylpyridinium chloride is preferably 259 nm; the mobile phase flow rate is 1 to 2 mL / min, specifically preferably 1 mL / min, 1.5 mL / min, 1.8 mL / min or 2 mL / min; the column temperature is 30 to 45°C, specifically preferably 30°C, 35°C, 40°C or 45°C; the injection volume is 20 to 100 μL, specifically preferably 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL or 100 μL.

[0040] In the present invention, when the easily ionizable compound is vecuronium bromide, the mobile phase of the high performance liquid chromatography analysis is preferably a mixed solvent of a first weakly alkaline tetramethylammonium hydroxide aqueous solution, methanol, acetonitrile and polyethylene glycol monomethyl ether; the volume ratio of the first weakly alkaline tetramethylammonium hydroxide aqueous solution, methanol and acetonitrile is preferably 120-150:235-265:600-630, more preferably 130-140:240-260:610-620, further preferably 135:250:615; the pH value of the first weakly alkaline tetramethylammonium hydroxide aqueous solution is preferably 6. The first weakly alkaline tetramethylammonium hydroxide aqueous solution is preferably obtained by adjusting the first tetramethylammonium hydroxide aqueous solution with acid, and the concentration of the first tetramethylammonium hydroxide aqueous solution is preferably 17 to 19 g / L, more preferably 17.5 to 18.5 g / L, and further preferably 18 g / L, based on tetramethylammonium hydroxide pentahydrate; the acid preferably includes phosphoric acid and / or glacial acetic acid.

[0041] In the present invention, when the easily ionizable compound is rocuronium bromide, the mobile phase of the high performance liquid chromatography analysis is preferably a mixed solvent of a second weak alkaline tetramethylammonium hydroxide solution, acetonitrile and polyethylene glycol monomethyl ether; the volume ratio of the second weak alkaline tetramethylammonium hydroxide solution to acetonitrile is preferably 5-15:85-95, more preferably 8-12:88-92, and further preferably 10:90; the pH value of the second weak alkaline tetramethylammonium hydroxide solution is preferably 6.5-8, more preferably 7-7.5, Specifically, it is preferably 6.5, 6.6, 6.7, 6.8, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8; the second weakly alkaline tetramethylammonium hydroxide aqueous solution is preferably obtained by adjusting the second tetramethylammonium hydroxide aqueous solution by phosphoric acid, and the concentration of the second tetramethylammonium hydroxide aqueous solution is preferably 0.02-0.03 mol / L, more preferably 0.022-0.028 mol / L, and further preferably 0.025 mol / L.

[0042] In the present invention, when the easily ionizable compound is cetylpyridinium chloride, the mobile phase of the high performance liquid chromatography analysis includes a mobile phase A and a mobile phase B; the mobile phase A is preferably methanol; the mobile phase B is preferably a mixed solution of a tetramethylammonium hydroxide aqueous solution (referred to as a third tetramethylammonium hydroxide aqueous solution), a potassium dihydrogen phosphate aqueous solution and polyethylene glycol monomethyl ether; the concentration of the third tetramethylammonium hydroxide aqueous solution is preferably 0.01 to 0.03 mol / L, more preferably 0.015 to 0.025 mol / L, and further preferably 0.02 mol / L; the pH of the mobile phase B is preferably 0.01 to 0.03 mol / L, more preferably 0.015 to 0.025 mol / L, and further preferably 0.02 mol / L. The value is preferably 3 to 4.5, more preferably 3.2 to 4.2, and further preferably 3.5; the pH value of the mobile phase B is preferably adjusted by an acid; the acid preferably includes phosphoric acid and / or glacial acetic acid; the elution method is preferably gradient elution, and the gradient elution program is preferably as follows: 0 to 8 min, the volume fraction of the mobile phase A increases from 50% to 70%; 8 to 30 min, the volume fraction of the mobile phase A is 70%; 30 to 32 min, the volume fraction of the mobile phase A decreases from 70% to 50%; 32 to 40 min, the volume fraction of the mobile phase A is 50%. In the present invention, the concentration of polyethylene glycol monomethyl ether in the mobile phase B is preferably 4 to 6 g / L, more preferably 4.5 to 5.5 g / L, and further preferably 5 g / L.

[0043] In the present invention, when the analysis is detection, the HPLC analysis obtains the detection result of the easily ionizable compound; when the analysis is separation, the HPLC analysis obtains the pure product of the easily ionizable compound.

[0044] The present invention also provides a method for regenerating a high performance liquid chromatography column, comprising the following steps: regenerating the chromatographic column used in the analytical method described in the above technical solution using a regenerated mobile phase; the regenerated mobile phase contains polyethylene glycol monomethyl ether. In the present invention, the regeneration is preferably: eluting using a regenerated mobile phase. In the present invention, the solvent is preferably a mobile phase; the concentration of polyethylene glycol monomethyl ether in the regenerated mobile phase is preferably 4 to 6 g / L, more preferably 4.5 to 5.5 g / L, and further preferably 5 g / L; the regenerated mobile phase is preferably the same as the mobile phase used in the analytical method for the easily ionized compound, which will not be repeated here. In the present invention, the elution time is preferably 4 to 8 hours, more preferably 5 to 7 hours, and further preferably 6 hours.

[0045] In order to further illustrate the present invention, the stabilizer and its application, the analysis method of easily ionized compounds and the regeneration method of the high performance liquid chromatography column provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0046] In the following examples, in vecuronium bromide for injection, impurity A is 2β,16β-di(piperidin-1-yl)-5α-androstane-3α,17β-diacetate diethyl ester; impurity C is 1-[17β-(acetyloxy)-3α-hydroxy-2β-(piperidin-1-yl)-5α-androstane-16β-yl]-1-methylpiperidin-1-yl; impurity D is 1-[3α,17β-dihydroxy-2β-(piperidin-1-yl)-5α-androstane-16β-yl]-1-methylpiperidin-1-yl; impurity E is 1-[3α-(acetyloxy)-17β-hydroxy-2β-(piperidin-1-yl)-5α-androstane-16β-yl]-1-methylpiperidin-1-yl.

[0047] In rocuronium bromide injection, impurity A: 3α,17β-(dihydroxy)-2β(morpholin-1-yl)-16-(pyrrolidin-1-yl)-5α-androst-17-acetate; impurity B: 1-allyl-l-[3α,17β-(dihydroxy)-2β-(morpholin-1-yl)-5α-androst-16β-yl]pyrrolidinium bromide-3,17-diacetate; impurity C: 1-allyl-1-[3β,17β(dihydroxy)-2β(morpholin-1-yl)-5a-androst-16β-yl]pyrrolidinium bromide; impurity D: 1-allyl-l-[3α-acetoxy,17β-hydroxy-2β-(morpholin-1-yl)-5α-androst-16β- Impurity E: 1-allyl-l-[3α-hydroxy,17β-acetoxy-2β-(pyridin-1-yl)-5α-androst-16β-yl]pyrrolidinium bromide; Impurity F: 1-allyl-l-[3α,17β-(dihydroxy)-2β-(pyrrolidinyl)-5β-androst-16β-yl]pyrrolidinium bromide-3,17-diacetate; Impurity G: 3α,17β-(dihydroxy)-2β(morpholin-1-yl)-16-(pyrrolidin-1-yl)-5α-androstane; Impurity H: 1-[2-(4-morpholinyl)-3-oxo-17β-acetoxy-5α-androst-1-en-16yl]-1-(2-propenyl)pyrrolidine.

[0048] Impurity I in cetylpyridinium chloride: 1-tetradecylpyridinium chloride.

[0049] The number average molecular weight of polyethylene glycol monomethyl ether is 500.

[0050] Example 1

[0051] Analysis of Vecuronium Bromide for Injection

[0052] (1.1) Perform high performance liquid chromatography analysis on vecuronium bromide for injection to obtain the test results of vecuronium bromide or pure vecuronium bromide. The analysis results are shown in Figure 1 and Table 1.

[0053] HPLC conditions:

[0054] Chromatographic column: Octadecylsilane bonded silica gel as filler (Thermo scientific ODS Hyoersil, 250×4.6mm, 5μm, unused new chromatographic column, chromatographic column number is recorded as No. 01); the chromatographic column was equilibrated with mobile phase for 4h before use;

[0055] Mobile phase: buffer (18.0 g / L tetramethylammonium hydroxide pentahydrate aqueous solution, pH adjusted to 8.0 with phosphoric acid): methanol: acetonitrile volume ratio = 135:250:615;

[0056] Detection wavelength: 210nm;

[0057] Mobile phase flow rate: 2 mL / min;

[0058] Column temperature: 40°C;

[0059] Injection volume: 20 μL.

[0060] Table 1 Separation results of the new chromatographic column for vecuronium bromide for injection

[0061] name Retention time (min) Separation Theoretical plates Tailing Factor Impurity C 4.217 -- 3364 0.979 Impurity D 4.659 1.537 4282 1.342 Vecuronium 5.517 1.843 1167 4.233 Impurity E 6.916 2.555 3792 1.396 Impurity A 20.531 24.356 15083 1.030

[0062] Depend on Figure 1 As shown in Table 1, the separation between the impurities and between the impurities and the main component (vecuronium bromide) peak is greater than 1.5, and the separation of each substance is high.

[0063] (1.2) Perform HPLC analysis on vecuronium bromide for injection according to the detection method of (1.1). The analysis results of vecuronium bromide for injection after the chromatographic column was used 30 times are shown in Figure 2 and Table 2.

[0064] Table 2 Separation results of vecuronium bromide for injection after the chromatographic column was used 30 times

[0065] name Retention time (min) Separation Theoretical plates Tailing Factor Impurity C 4.223 -- 4827 1.117 Impurity D 5.140 3.450 5073 1.181 Vecuronium 5.537 0.828 1089 4.857 Impurity E 8.052 3.894 2697 1.357 Impurity A 19.866 18.831 15682 1.043

[0066] Depend on Figure 1-2 As shown in Tables 1 and 2, after the chromatographic column has been used for a period of time, the retention time of the impurity peaks is delayed, and the delay trend of the impurity D and impurity E peak retention time is more obvious, which leads to an increase in the peak separation between impurity D and impurity C, and a decrease in the peak separation between impurity D and the main component to 0.828, which is less than 1.5 and does not meet the separation requirements, while the peak separation between impurity E and the main component increases. This shows that under the same chromatographic conditions, the separation of impurities will change with the number of times the chromatographic column is used.

[0067] (1.3) Perform HPLC analysis on vecuronium bromide for injection according to the detection method of (1.1). The only difference from (1.1) is that the buffer solution pH is 7.5. The analysis results of vecuronium bromide for injection are shown in Figure 3 and Table 3.

[0068] Table 3 Separation results of the new chromatographic column for vecuronium bromide for injection

[0069] name Retention time (min) Separation Theoretical plates Tailing Factor Impurity C 4.804 -- 8077 1.348 Impurity D 5.403 2.373 5517 1.895 Vecuronium 6.302 1.880 1432 4.052 Impurity E 7.950 2.936 4857 1.350 Impurity A 19.343 21.135 15479 0.993

[0070] Depend on Figure 3 As shown in Table 3, after adjusting the pH value of the mobile phase, the peak separation between impurity D and the main component increased, and the peak separation between each impurity and between the impurity and the main component reached above 1.5, achieving a high degree of separation between each impurity and between the impurity and the main component.

[0071] (1.4) Perform HPLC analysis on vecuronium bromide for injection according to the detection method of (1.3). The analysis results of vecuronium bromide for injection after the chromatographic column was used 20 times are shown in Figure 4 and Table 4.

[0072] Table 4 Separation results of vecuronium bromide for injection after the chromatographic column was used 20 times

[0073] name Retention time (min) Separation Theoretical plates Tailing Factor Peak to Valley Ratio Impurity C 4.566 -- 6922 1.117 -- Impurity D 5.348 2.661 3375 1.523 -- Vecuronium 6.058 1.347 1249 4.059 -- Impurity E 8.284 2.991 1702 -- 2.737 Impurity A 20.844 16.959 15124 0.995 --

[0074] Note: “--” means there is no data or no statistics are needed.

[0075] Depend on Figure 3-4 As shown in Tables 3 and 4, under the same chromatographic conditions, after the same chromatographic column has been used for a period of time, the peak separation between impurity D and impurity C increases, the peak separation between impurity D and the main component decreases, and the peak separation between impurity E and the main component increases. This shows that under the same chromatographic conditions, the separation between impurities will change with the number of times the chromatographic column is used. When the chromatographic column is used 20 times, the separation between impurity D and the main component is less than 1.5, and the separation effect of the chromatographic column deteriorates.

[0076] (1.5) The vecuronium bromide for injection was analyzed by high performance liquid chromatography according to the detection method of (1.1). The only difference from (1.1) was that the batch of chromatographic columns was different (the chromatographic column number was recorded as No. 02). The analysis results of vecuronium bromide for injection after the chromatographic column was used 30 times were shown in Figure 5 and Table 5.

[0077] Table 5 Separation results of vecuronium bromide for injection after the chromatographic column was used 30 times

[0078] name Retention time (min) Separation Theoretical plates Tailing Factor Peak to Valley Ratio Impurity C 4.756 -- 4005 1.079 -- Impurity D 5.514 2.332 3984 -- 109.168 Vecuronium 6.294 1.513 1364 3.927 -- Impurity E 8.305 3.215 3401 -- 6.000 Impurity A 19.399 18.635 15613 1.007 --

[0079] Note: “--” means there is no data or no statistics are needed.

[0080] Depend on Figure 2 , Figure 5 It can be seen from Table 2 and Table 5 that when the same chromatographic conditions and the same model of chromatographic columns from different batches are used the same number of times, the separation effects between impurities and between impurities and main components are different, indicating that the batch differences of chromatographic columns have an impact on the separation of impurities.

[0081] (1.6) The vecuronium bromide for injection was analyzed by high performance liquid chromatography according to the detection method of Example 1. The only difference from Example 1 is that the mobile phase: buffer (18.0 g / L aqueous solution of tetramethylammonium hydroxide pentahydrate, pH=8.0 adjusted with phosphoric acid): methanol: acetonitrile volume ratio = 135:250:615, and the mobile phase also contains 5 g / L polyethylene glycol monomethyl ether. The analysis results of vecuronium bromide for injection after the chromatographic column was used 60 times are shown in Figure 6 and Table 6.

[0082] Table 6 Separation results of vecuronium bromide for injection after the chromatographic column was used 60 times

[0083] name Retention time (min) Separation Theoretical plates Tailing Factor Impurity C 4.440 -- 6361 1.245 Impurity D 4.939 1.988 5021 1.455 Vecuronium 5.837 1.936 1293 4.189 Impurity E 7.268 2.636 4441 1.420 Impurity A 20.431 24.294 15938 1.022

[0084] Note: “--” means there is no data or no statistics are needed.

[0085] Depend on Figure 4 , Figure 6 It can be seen from Table 4 and Table 6 that after adding polyethylene glycol monomethyl ether to the mobile phase and controlling the pH value of the mobile phase, the service life of the chromatographic column is significantly increased.

[0086] Example 2

[0087] Analysis of Rocuronium Bromide Injection

[0088] (2.1) Perform high performance liquid chromatography analysis on rocuronium bromide injection to obtain the test results of rocuronium bromide or pure rocuronium bromide. The analysis results are shown in Figure 7 and Table 7.

[0089] HPLC conditions:

[0090] Chromatographic column: silica gel as filler (HALO Hilic, 4.6 mm × 250 mm, 5 μm, unused new chromatographic column, the chromatographic column number is recorded as No. 02);

[0091] Before using the chromatographic column, perform routine activation and balance: flush with methanol at a flow rate of 0.2 mL / min for 30 min, increase the flow rate from 0.2 mL / min to 1 mL / min for 90 min, and then balance with mobile phase for 4 h;

[0092] Mobile phase: 0.025 mol / L tetramethylammonium hydroxide aqueous solution (4.53 g of tetramethylammonium hydroxide pentahydrate, add 1000 mL of water, shake well, and adjust the pH value to 7.4 with phosphoric acid): acetonitrile volume ratio = 10:90.

[0093] Detection wavelength: 210nm;

[0094] Mobile phase flow rate: 2.0 mL / min;

[0095] Column temperature: 30°C;

[0096] Injection volume: 10 μL.

[0097] Table 7 Separation results of the new chromatographic column for rocuronium bromide injection

[0098] name Relative retention time Retention time (min) Separation Impurity A 0.22 1.379 -- Impurity G 0.58 3.651 18.583 Impurity F 0.69 4.286 4.297 Impurity B 0.75 4.694 2.696 Impurity D 0.84 5.259 3.535 Impurity H 0.93 5.812 3.209 Rocuronium 1.00 6.254 2.442 Impurity C 1.18 7.370 5.708 Impurity E 1.85 11.576 14.773

[0099] Depend on Figure 7 As can be seen from Table 7, after the new chromatographic column undergoes conventional activation and equilibrium, the separation between impurities in rocuronium bromide injection meets the baseline separation, and the separation between impurity C and rocuronium bromide meets the separation requirement of 3.5 under the rocuronium variety item of the 2020 edition (Part II) of the "Chinese Pharmacopoeia".

[0100] (2.2) The rocuronium bromide injection was subjected to HPLC analysis according to the detection method of (2.1). The separation results of vecuronium bromide for injection after the chromatographic column was used 20 times were shown in Figure 8 and Table 8.

[0101] Table 8 Separation results of rocuronium bromide injection after the chromatographic column was used 20 times

[0102]

[0103]

[0104] Depend on Figure 7-Figure 8 As shown in Tables 7 and 8, after 20 injections of the chromatographic column, the separation between impurity H and impurity D, and the separation between rocuronium bromide and impurity H in rocuronium bromide injection showed a decreasing trend.

[0105] (2.3) The chromatographic column used 20 times in (2.2) was used to perform HPLC analysis on rocuronium bromide injection. The only difference from (2.1) was that the pH of the tetramethylammonium hydroxide aqueous solution was 7.8, and the mobile phase also contained 5 g / L of polyethylene glycol monomethyl ether. The separation results of rocuronium bromide injection are shown in Fig. 9 and Table 9.

[0106] Table 9 Separation results of rocuronium bromide injection by chromatographic column

[0107] name Relative retention time Retention time (min) Separation Impurity A 0.22 1.427 / Impurity G 0.50 3.320 15.847 Impurity F 0.69 4.556 8.249 Impurity B 0.75 5.009 2.875 Impurity D 0.85 5.653 3.841 Impurity H 0.93 6.210 3.108 Rocuronium 1.00 6.636 2.211 Impurity C 1.19 7.919 6.027 Impurity E 1.62 10.744 10.264

[0108] Depend on Figure 8 , Fig. 9 As shown in Tables 8 and 9, after adding polyethylene glycol monomethyl ether and controlling the pH value of the mobile phase, the separation between impurity H and impurity D, and the separation between rocuronium bromide and impurity H were improved.

[0109] Example 3

[0110] Analysis of Cetylpyridinium Chloride

[0111] (3.1) Cetylpyridinium chloride is subjected to high performance liquid chromatography analysis to obtain the detection result of cetylpyridinium chloride or the pure product of cetylpyridinium chloride. The analysis results are shown in Table 11.

[0112] HPLC conditions:

[0113] Chromatographic column: cyano-bonded silica gel as filler (Ultimate XB-CN, 250×4.6 mm, 5 μm, unused new chromatographic column, chromatographic column number is recorded as No. 03);

[0114] Before using the chromatographic column, perform routine activation and balance: flush with methanol at a flow rate of 0.2 mL / min for 30 min, increase the flow rate from 0.2 mL / min to 1 mL / min for 90 min, and then balance with mobile phase for 4 h;

[0115] Mobile phase A: methanol;

[0116] Mobile phase B: 0.02 mol / L tetramethylammonium hydroxide aqueous solution: 0.003 mol / L potassium dihydrogen phosphate aqueous solution, volume ratio = 10:3, and acetic acid was used to adjust the pH of mobile phase B to 3.5 ± 0.05;

[0117] Gradient elution program: see Table 10.

[0118] Detection wavelength: 259nm;

[0119] Mobile phase flow rate: 1 mL / min;

[0120] Column temperature: 30°C;

[0121] Injection volume: 50 μL.

[0122] Table 10 Gradient elution program

[0123] Time (min) Mobile phase A volume fraction (%) Volume fraction of mobile phase B (%) 0 50 50 8 70 30 30 70 30 32 50 50 40 50 50

[0124] Table 11 Separation results of new chromatographic column for cetylpyridinium chloride

[0125] name Relative retention time Retention time (min) Separation Impurity I 0.65 6.833 / Cetylpyridinium chloride 1.0 10.513 4.825

[0126] It can be seen from Table 11 that after the new chromatographic column undergoes conventional activation equilibrium, the separation between impurities basically meets the baseline separation, and the separation between impurity I and cetylpyridinium chloride meets the separation requirements under the cetylpyridinium chloride variety item of the 2020 edition (Part II) of the "Chinese Pharmacopoeia" (meets the requirements and is considered to be not less than 1.5).

[0127] (3.2) Cetylpyridinium chloride was analyzed by HPLC according to the detection method of (3.1). The separation results of cetylpyridinium chloride after the chromatographic column was used 50 times are shown in Table 12.

[0128] Table 12 Separation results of cetylpyridinium chloride after the chromatographic column was used 50 times

[0129] name Relative retention time Retention time (min) Separation Impurity I 0.70 8.281 / Cetylpyridinium chloride 1.0 11.832 4.316

[0130] It can be seen from Tables 11 and 12 that after 50 injections of the chromatographic column, the separation between impurity I and cetylpyridinium chloride showed a decreasing trend.

[0131] (3.3) Cetylpyridinium chloride was analyzed by HPLC according to the detection method of (3.1). The only difference from (3.1) was that the mobile phase B also contained 5 g / L of polyethylene glycol monomethyl ether, and the chromatographic column only needed to be equilibrated with the mobile phase for 4 h before use. The separation results of cetylpyridinium chloride are shown in Table 13.

[0132] Table 13 Separation results of cetylpyridinium chloride after using the chromatographic column (No. 04) 50 times

[0133] name Relative retention time Retention time (min) Separation Impurity I 0.68 7.390 / Cetylpyridinium chloride 1.0 10.869 4.519

[0134] It can be seen from Tables 12 and 13 that after adding polyethylene glycol monomethyl ether and controlling the pH value of the mobile phase, the separation between impurity I and cetylpyridinium chloride increased from 4.316 to 4.519, and the separation was improved.

[0135] In summary, in the present invention, in the chromatography for separating ionic compounds using ion pair reagents, by adding polyethylene glycol monomethyl ether to the mobile phase and adjusting and controlling the pH value in the mobile phase, the chromatographic column ((1.6) in Example 1) that has been unable to meet the separation requirements can meet the chromatographic peak separation requirements again.

[0136] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A stabilizer comprising polyethylene glycol monomethyl ether and a pH regulator.

2. Use of the stabilizer according to claim 1 in liquid chromatography analysis of easily ionizable compounds; the analysis is detection or separation.

3. The use according to claim 2, characterized in that: The easily ionizable compound includes one or more of vecuronium bromide, rocuronium bromide, pipecuronium bromide and cetylpyridinium chloride.

4. A method for analyzing easily ionizable compounds, comprising the following steps: Analyzing the easily ionizable compound to be tested to obtain an analysis result of the easily ionizable compound; The analysis is detection or separation; The mobile phase used in the high performance liquid chromatography analysis contains the stabilizer according to claim 1.

5. The analysis method according to claim 4, characterized in that The concentration of polyethylene glycol monomethyl ether in the mobile phase is 4-6 g / L.

6. The analysis method according to claim 4, characterized in that The conditions for the high performance liquid chromatography analysis include: the filler of the chromatographic column includes octadecylsilane bonded silica gel, silica gel or cyano bonded silica gel, the detection wavelength is 210-260nm, the mobile phase flow rate is 1-2mL / min, the column temperature is 30-45°C, and the injection volume is 20-100μL.

7. The analysis method according to claim 5, characterized in that When the easily ionizable compound is vecuronium bromide, the mobile phase of the high performance liquid chromatography analysis is a mixed solvent of a first weakly alkaline tetramethylammonium hydroxide aqueous solution, methanol, acetonitrile and polyethylene glycol monomethyl ether; the volume ratio of the first weakly alkaline tetramethylammonium hydroxide aqueous solution, methanol and acetonitrile is 120-150:235-265:600-630; the pH value of the first weakly alkaline tetramethylammonium hydroxide aqueous solution is 6.5-8.

8. The analysis method according to claim 5, characterized in that When the easily ionizable compound is rocuronium bromide, the mobile phase of the high performance liquid chromatography analysis is a mixed solvent of a second weak alkaline tetramethylammonium hydroxide solution, acetonitrile and polyethylene glycol monomethyl ether; the volume ratio of the second weak alkaline tetramethylammonium hydroxide solution to acetonitrile is 5-15:85-95; the pH value of the second weak alkaline tetramethylammonium hydroxide solution is 6.5-8.

9. The analysis method according to claim 5, characterized in that When the easily ionizable compound is cetylpyridinium chloride, the mobile phase of the high performance liquid chromatography analysis includes mobile phase A and mobile phase B; the mobile phase A is methanol; the mobile phase B is a mixed solution of tetramethylammonium hydroxide aqueous solution, potassium dihydrogen phosphate aqueous solution and polyethylene glycol monomethyl ether; the pH value of the mobile phase B is 3-4.5; the elution method is gradient elution, and the gradient elution program is as follows: from 0 to 8 minutes, the volume fraction of the mobile phase A increases from 50% to 70%; from 8 to 30 minutes, the volume fraction of the mobile phase A is 70%; from 30 to 32 minutes, the volume fraction of the mobile phase A decreases from 70% to 50%; from 32 to 40 minutes, the volume fraction of the mobile phase A is 50%.

10. A method for regenerating a high performance liquid chromatography column, comprising the following steps: The chromatographic column used in the analytical method according to any one of claims 4 to 9 is regenerated by using a regenerated mobile phase; the regenerated mobile phase contains polyethylene glycol monomethyl ether.

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