Method for simultaneously detecting sodium chloride and 4-hydroxyethylpiperazine ethanesulfonic acid and application

Abstract

The invention discloses a method for simultaneously detecting sodium chloride and 4-hydroxyethylpiperazine ethanesulfonic acid and application thereof.According to the method, reversed-phase chromatography is combined with an electrospray detector, sodium chloride and 4-hydroxyethylpiperazine ethanesulfonic acid in a preparation can be detected under the same chromatographic condition, and the method is easy and rapid to operate, high in sensitivity, high in specificity and good in repeatability; the method is suitable for analyzing other products with similar auxiliary materials.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis technology, and in particular to a high performance liquid phase analysis method for simultaneously detecting sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid. Background Art

[0002] As pre-market drug review requirements for key excipients in preparations become increasingly stringent, quantitative analysis of commonly used excipients has received increasing attention in the quality research of preparations.

[0003] Irinotecan hydrochloride liposome injection is a nanoliposome PEGylated preparation containing irinotecan developed and launched by Ipsen. It has been approved for marketing in the United States, Germany, Japan, China and other countries and regions. The commercially available irinotecan liposome injection contains 4.05 mg of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) as a buffer and 8.42 mg of sodium chloride as an isotonic agent per mL. HEPES and sodium chloride are two key excipients. Although sodium chloride has ultraviolet absorption, it has poor retention on ordinary chromatographic columns. HEPES has no ultraviolet absorption. The two substances cannot be well separated, leaving a gap in the quality research of key excipients in the same field. Summary of the invention

[0004] In view of this, the present application provides a method for simultaneously detecting sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES). Specifically, a high performance liquid analysis method is used to detect the content of excipients sodium chloride and HEPES in a preparation, especially irinotecan liposome injection. The method is simple to operate, quick to analyze, and has good stability, specificity, and durability.

[0005] In order to achieve the above-mentioned purpose of the invention, the present application provides a method for simultaneously detecting multiple excipients in a preparation, which adopts high performance liquid chromatography for detection.

[0006] In an exemplary embodiment, the preparation is a liposome injection, preferably, the preparation is irinotecan hydrochloride liposome injection.

[0007] In an exemplary embodiment, the auxiliary materials are sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES).

[0008] In an exemplary embodiment, the method comprises the following steps:

[0009] S1. Solution preparation:

[0010] Prepare mobile phase;

[0011] Prepare reference solution;

[0012] Prepare test sample solution;

[0013] S2. Detection:

[0014] The reference solution and the test solution are injected into a high performance liquid chromatograph for detection, and the chromatogram is recorded.

[0015] In an exemplary embodiment, in step S2, in the high performance liquid chromatography detection, the mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of a volatile salt, preferably an aqueous solution of ammonium formate or ammonium acetate; mobile phase B is an organic solvent, optionally methanol or acetonitrile, preferably acetonitrile.

[0016] In an exemplary embodiment, the volume ratio of the mobile phase A to the mobile phase B is (25:75)-(20:80); preferably, 25:75, 24:76, 23:77, 22:78, 21:79 or 20:80.

[0017] In an exemplary embodiment, the concentration of the mobile phase A is 50 mM-75 mM; preferably, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM or 75 mM.

[0018] In an exemplary embodiment, the pH value of the mobile phase A is 2.5-3.5; preferably, 2.5-3.0 or 3.0-3.5; more preferably, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5.

[0019] In an exemplary embodiment, optionally, the pH adjuster of the mobile phase A is formic acid or acetic acid.

[0020] In an exemplary embodiment, in step S2, other chromatographic conditions include:

[0021] Chromatographic column: Optionally, a bonded hydrophilic chromatographic column;

[0022] Chromatographic column model: Optionally, a Merck SeQuant ZIC-chilic (100×4.6 mm, 3 μm) chromatographic column, or a Pheromones Comixsl HCS (150×3.0 mm, 3 μm) chromatographic column;

[0023] Flow rate: optionally, 0.8 mL / min-1.2 mL / min, preferably, 0.8 mL / min-1.0 mL / min or 1.0 mL / min-1.2 mL / min, more preferably, 0.8 mL / min, 1.0 mL / min or 1.2 mL / min;

[0024] Elution mode: isocratic elution;

[0025] The temperature of the chromatographic column: optionally, 25°C-40°C, preferably, 25°C-30°C, 30°C-35°C or 35°C-40°C, more preferably, 25°C, 30°C, 35°C or 40°C;

[0026] Injection volume: optionally, 10 μL-50 μL, preferably, 20 μL.

[0027] Detector: Charged aerosol (CAD) detector.

[0028] In an exemplary embodiment, the detector condition is:

[0029] Atomization temperature: optionally, 35°C or 55°C;

[0030] Data acquisition frequency: optionally, 2 Hz-10 Hz, preferably, 2 Hz-5 Hz or 5 Hz-10 Hz, more preferably, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz or 10 Hz;

[0031] Filter constant: optionally, 2.0s-10.0s, preferably, 2s-5s or 5s-10s, more preferably, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s or 10s.

[0032] In an exemplary embodiment, in step S1, the preparation method of the mobile phase is as follows:

[0033] Ammonium formate or ammonium acetate is added with water to prepare mobile phase A;

[0034] Adjust the pH of mobile phase A;

[0035] The mobile phase A and the mobile phase B are mixed to prepare a mobile phase mixture.

[0036] In an exemplary embodiment, in step S1, the preparation method of the reference substance solution is as follows: sodium chloride and HEPES standard substance plus solubilizing agent are placed in the same volumetric flask and dissolved to prepare a reference substance stock solution; the reference substance stock solution is diluted to obtain the reference substance solution.

[0037] In an exemplary embodiment, optionally, in the reference solution, the concentrations of sodium chloride and HEPES are: 1.611 μg / ml-0.74 mg / ml sodium chloride, 4.025 μg / ml-0.36 mg / ml HEPES; preferably, 163 μg / ml sodium chloride and 81 μg / ml HEPES.

[0038] In an exemplary embodiment, in step S1, the method for preparing the test solution is as follows: a test sample containing sodium chloride and a HEPES preparation is dissolved in a solvent to obtain a test solution.

[0039] In an exemplary embodiment, in step S1, the solvent of the reference solution and the test solution is a methanol aqueous solution with a volume percentage of 90%.

[0040] In an exemplary embodiment, the method further includes:

[0041] S3. Analysis:

[0042] According to the chromatograms of the reference solution and the test solution, the contents of sodium chloride and HEPES in the test sample were calculated by the external standard method.

[0043] In an exemplary embodiment, in step S3, the calculation formula for the content of sodium chloride and HEPES in the test sample is as follows:

[0044]

[0045] The method of the present application fills the gap in the prior art, and the method is simple and fast to operate, has good stability and specificity, and provides a simple and reliable analytical method for the original research prescription analysis and quality research of irinotecan liposome injection.

[0046] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0048] Figure 1 is the HPLC chromatogram of the solvent in Example 1;

[0049] Figure 2-Figure 5 They are respectively the high performance liquid chromatograms of chromatographic columns 1-4 in Example 1;

[0050] Figure 6-Figure 8 They are respectively the HPLC chromatograms of mobile phases 2-4 in Example 2;

[0051] Figure 9-10 They are respectively the HPLC chromatograms of mobile phases 5-6 in Example 3;

[0052] Figure 11-Figure 12 They are respectively the HPLC chromatograms of mobile phases 7-8 in Example 4;

[0053] Figure 13-14 They are respectively the HPLC chromatograms of mobile phases 9-10 in Example 5;

[0054] Figure 15-17 They are respectively the HPLC chromatograms at column temperatures of 25°C, 35°C, and 40°C in Example 6;

[0055] Figure 18-19 They are respectively the HPLC chromatograms when the mobile phase flow rates are 0.8 mL / min and 1.2 mL / min in Example 7;

[0056] Fig. 20 This is the HPLC chromatogram with the nebulization temperature of the CAD detector being 55° C. in Example 8;

[0057] Figure 21-22 They are respectively the HPLC chromatograms when the data acquisition frequency of the CAD detector in Example 9 is 2 Hz and 10 Hz;

[0058] Figure 23-Figure 24 They are respectively the HPLC chromatograms when the filter constants of the CAD detector in Example 10 are 2s and 5s;

[0059] Figure 25-26 They are respectively the HPLC chromatograms when the injection volumes are 10 μL and 50 μL in Example 11;

[0060] Figure 27-Figure 28 are linear relationship diagrams of sodium chloride and HEPES in Example 12, respectively;

[0061] Figure 29-Figure 32 They are respectively the HPLC chromatograms of different batches of reference preparation and self-made product in Example 13. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solution and advantages of the present application more clear, the embodiments of the present invention will be described in detail below. It should be noted that the embodiments and features in the embodiments of the present application can be combined with each other arbitrarily without conflict.

[0063] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0064] Example 1 Investigation of chromatographic column

[0065] S1. Solution preparation:

[0066] Preparation of mobile phase 1: dilute ammonium formate with water to prepare mobile phase A1 with a concentration of 50 mM, and use formic acid to adjust the pH of mobile phase A1 to 3.0; take mobile phase A1 and acetonitrile (mobile phase B) and prepare mobile phase 1 with a volume ratio of mobile phase A1:mobile phase B=20:80.

[0067] Preparation of reference solution: Sodium chloride and HEPES standards were ultrasonically dissolved in a 90% by volume methanol aqueous solution (solvent) to prepare a mixed solution containing 163 μg sodium chloride and 81 μg HEPES per mL.

[0068] S2. Chromatographic detection:

[0069] The effects of the following four chromatographic columns on the separation ability and retention time of sodium chloride and HEPES were investigated.

[0070] Measure 20 μL of the above-mentioned reference solution and inject it into the high-performance liquid chromatograph to record the chromatogram; measure 20 μL of the above-mentioned solvent and inject it into the high-performance liquid chromatograph, and use the following chromatographic column 1 to record the chromatogram of the solvent. Among them, the main technical parameters of the high-performance liquid chromatograph are as follows:

[0071] Instrument: Dinoex U3000;

[0072] Column 1: Merck SeQuant ZIC-chilic (100×4.6 mm, 3 μm);

[0073] Column 2: Waters Atlantis T3 (150×4.6 mm, 3 μm);

[0074] Column 3: YMC-Triart C18 (150×4.6mm, 3μm)

[0075] Chromatographic column 4: Phenomenon Comixsl HCS (150×3.0mm, 3μm)

[0076] Column temperature: 30°C;

[0077] Flow rate: 1.0 mL / min;

[0078] Injection volume: 20 μL;

[0079] CAD detector: data acquisition frequency: 5 Hz; atomizer temperature: 35°C; filter constant: 10.0 s.

[0080] S3. Chromatogram analysis:

[0081] The chromatogram of the solvent is Figure 1 shown.

[0082] The chromatograms corresponding to columns 1, 2, 3, and 4 are as follows: Figure 2 , 3 , 4, 5.

[0083] according to Figure 2 It can be seen that when using chromatographic column 1, the chromatographic peak with a retention time of 5.5 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.2 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.7 min is the HEPES peak; according to Figure 3-Figure 4 It can be seen that when using chromatographic columns 2 and 3, the ordinary hydrophilic column (chromatographic column 2) and the ordinary C18 column (chromatographic column 3) have weak retention for sodium chloride and HEPES; Figure 5 It can be seen that when chromatographic column 4 is used, the chromatographic peak with a retention time of 0.786 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 5.794 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 4.358 min is the HEPES peak.

[0084] Therefore, the present application can use chromatographic column 1 and chromatographic column 4 to detect and separate sodium chloride and HEPES.

[0085] Example 2: Investigation of mobile phase A

[0086] S1. Solution preparation:

[0087] Preparation of mobile phases 2, 3, and 4: Weigh ammonium acetate, formic acid, and acetic acid respectively, and dilute with water to prepare mobile phase A2, A3, and A4 solutions with a concentration of 50 mM. Use acetic acid to adjust the pH of mobile phase A2 to 3.0. Take mobile phase A2, A3, and A4 solutions and acetonitrile (mobile phase B) and prepare mobile phases 2, 3, and 4 with a volume ratio of mobile phase A2, A3, and A4: mobile phase B = 20:80.

[0088] The preparation method of the reference solution is the same as that in Example 1.

[0089] S2. Chromatographic detection:

[0090] Chromatographic column 1 was used for detection to examine the effects of different mobile phase compositions on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as in Example 1.

[0091] S3. Chromatogram analysis:

[0092] The chromatograms corresponding to mobile phases 2, 3, and 4 are as follows: Figure 6 , 7 、8.

[0093] according to Figure 6It can be seen that when mobile phase 2 is used as the mobile phase, the chromatographic peak with a retention time of 5.9 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.0 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 11.4 min is the HEPES peak; according to Figure 7-8 It can be seen that when mobile phases 3 and 4 were used as mobile phases, the three components did not produce normal peaks.

[0094] Therefore, the mobile phase A in the present application can be an aqueous solution of ammonium formate or ammonium acetate.

[0095] Example 3: Investigation of the volume ratio of mobile phases A and B

[0096] S1. Solution preparation:

[0097] Preparation of mobile phases 5 and 6: dilute ammonium formate with water to prepare mobile phase A5 and A6 solutions with a concentration of 50 mM, adjust the pH to 3.0 with formic acid, take mobile phase A5 and A6 solutions and acetonitrile (mobile phase B), and prepare mobile phase 5 with a volume ratio of mobile phase A5:mobile phase B = 25:75, and mobile phase 6 with a volume ratio of mobile phase A6:mobile phase B = 22:78.

[0098] The preparation method of the reference solution is the same as that in Example 1.

[0099] S2. Chromatographic detection:

[0100] Chromatographic column 1 was used for detection to examine the effects of different mobile phase compositions on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as in Example 1.

[0101] S3. Chromatogram analysis:

[0102] The chromatograms corresponding to mobile phases 5 and 6 are as follows: Figure 9-10 .

[0103] according to Fig. 9 It can be seen that when mobile phase 5 is used as the mobile phase, the chromatographic peak with a retention time of 3.9 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 5.4 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 5.9 min is the HEPES peak; according to Fig.10 It can be seen that when mobile phase 6 is used as the mobile phase, the chromatographic peak with a retention time of 4.7 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 6.8 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 8.2 min is the HEPES peak.

[0104] Example 4: Investigation of mobile phase A concentration

[0105] S1. Solution preparation:

[0106] Preparation of mobile phases 7 and 8: dilute ammonium formate with water to prepare mobile phase A7 and A8 solutions with concentrations of 25 mM and 75 mM, respectively, adjust the pH to 3.0 with formic acid, take mobile phase A7, A8 solutions and acetonitrile (mobile phase B), and prepare mobile phases 7 and 8 with a volume ratio of mobile phase A7, A8: mobile phase B = 20:80.

[0107] The preparation method of the reference solution is the same as that in Example 1.

[0108] S2. Chromatographic detection:

[0109] Chromatographic column 1 was used for detection to examine the effects of different mobile phase compositions on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as in Example 1.

[0110] S3. Chromatogram analysis:

[0111] The chromatograms corresponding to mobile phases 7 and 8 are as follows: Fig.11 , 12 .

[0112] according to Fig.11 It can be seen that when mobile phase 7 is used as the mobile phase, the three components do not produce peaks normally; Fig.12 It can be seen that when mobile phase 8 is used as the mobile phase, the chromatographic peak with a retention time of 5.6 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 7.7 min is the sodium ion peak of sodium chloride, the chromatographic peak with a retention time of 11.0 min is the HEPES peak, and when the ammonium formate concentration is 75 mM, it has no significant effect on the peak response and separation of each component.

[0113] Example 5: Investigation of pH value of mobile phase A

[0114] S1. Solution preparation:

[0115] Preparation of mobile phases 9 and 10: dilute ammonium formate with water to prepare mobile phases A9 and A10 solutions with a concentration of 50 mM, use formic acid to adjust the pH of the solutions to 2.5 and 3.5, respectively, take mobile phases A9 and A10 solutions and acetonitrile (mobile phase B), and prepare mobile phases 9 and 10 with a volume ratio of mobile phase A9, A10: mobile phase B = 20:80.

[0116] The preparation method of the reference solution is the same as that in Example 1.

[0117] S2. Chromatographic detection:

[0118] The chromatographic column 1 was used for detection to investigate the effects of different pH values ​​of the mobile phase A on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as those in Example 1.

[0119] S3. Chromatogram analysis:

[0120] The chromatograms corresponding to mobile phases 9 and 10 are as follows: Fig.13 , 14 .

[0121] according to Fig.13 As shown, when mobile phase 9 (the lowest pH tolerance limit near the chromatographic column is 2.0) is used as the mobile phase, the chromatographic peak with a retention time of 5.3 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.6 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.6 min is the HEPES peak; according to Fig.14 As shown, when mobile phase 10 is used as the mobile phase, the chromatographic peak with a retention time of 5.7 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 7.5 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 11.2 min is the HEPES peak. At this time, the separation between sodium ions and HEPES becomes smaller, and the separation becomes worse, which can indicate that when the pH is higher than this limit, the separation is worse.

[0122] Example 6: Investigation of column temperature

[0123] S1. Solution preparation:

[0124] The preparation methods of mobile phase and reference solution are the same as those in Example 1.

[0125] S2. Chromatographic detection:

[0126] Chromatographic column 1 was used and the temperature of the chromatographic column was set to 25° C., 35° C. and 40° C. respectively, to investigate the effects of different column temperatures on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as in Example 1.

[0127] S3. Chromatogram analysis:

[0128] The chromatograms corresponding to column temperatures of 25°C, 35°C, and 40°C are as follows: Fig.15 , 16 、17.

[0129] according to Fig.15 As shown, when the column temperature is 25°C, the chromatographic peak with a retention time of 5.5 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.4 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 11.1 min is the HEPES peak; according to Fig.16 As shown, when the column temperature is 35°C, the chromatographic peak with a retention time of 5.4 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.0 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.3 min is the HEPES peak; according to Fig.17As shown, when the column temperature is 40°C, the chromatographic peak with a retention time of 5.4 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 7.8 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.0 min is the HEPES peak.

[0130] Example 7: Investigation of mobile phase flow rate

[0131] S1. Solution preparation:

[0132] The preparation methods of mobile phase and reference solution are the same as those in Example 1.

[0133] S2. Chromatographic detection:

[0134] The flow rates of the mobile phase were set to 0.8 mL / min and 1.2 mL / min, respectively, and the chromatographic column 1 was used for detection to investigate the effects of different flow rates on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as in Example 1.

[0135] S3. Chromatogram analysis:

[0136] The corresponding chromatograms when the mobile phase flow rate is 0.8mL / min and 1.2mL / min are as follows: Fig.18 , 19 .

[0137] according to Fig.18 As shown in the figure, when the flow rate of the mobile phase is 0.8 mL / min, the chromatographic peak with a retention time of 6.8 min is the chloride ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.3 min is the sodium ion peak of sodium chloride. HEPES did not produce a peak within the running time, but was delayed to after 12 min. Fig.19 As shown, when the flow rate of the mobile phase is 1.2 mL / min, the chromatographic peak with a retention time of 4.5 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 6.8 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 8.9 min is the HEPES peak.

[0138] Example 8: Investigation of the atomization temperature of the detector

[0139] S1. Solution preparation:

[0140] The preparation methods of mobile phase and reference solution are the same as those in Example 1.

[0141] S2. Chromatographic detection:

[0142] The temperature of the nebulizer was set to 55° C., and the chromatographic column 1 was used for detection to investigate the effect of the temperature of the nebulizer on the separation ability and retention time of sodium chloride and HEPES. The other chromatographic conditions were the same as those in Example 1.

[0143] S3. Chromatogram analysis:

[0144] When the nebulizer temperature is 55°C, the corresponding chromatogram is: Fig. 20 .

[0145] according to Fig. 20 As shown, when the nebulizer temperature is 55°C, the chromatographic peak with a retention time of 5.5 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.2 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.7 min is the HEPES peak.

[0146] Example 9: Investigation of detector data acquisition frequency

[0147] S1. Solution preparation:

[0148] The preparation methods of mobile phase and reference solution are the same as those in Example 1.

[0149] S2. Chromatographic detection:

[0150] The data acquisition frequency of the CAD detector was set to 2 Hz and 10 Hz, and the chromatographic column 1 was used for detection to investigate the effects of different detector data acquisition frequencies on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as in Example 1.

[0151] S3. Chromatogram analysis:

[0152] The corresponding chromatograms when the CAD detector data acquisition frequency is 2Hz and 10Hz are Fig.21 , 22 .

[0153] according to Fig.21 As shown, when the data acquisition frequency is 2 Hz, the chromatographic peak with a retention time of 5.5 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.2 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.7 min is the HEPES peak; according to Fig. 22 As shown, when the data acquisition frequency is 10 Hz, the chromatographic peak with a retention time of 5.5 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.2 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.7 min is the HEPES peak.

[0154] Therefore, the data acquisition frequency range of the detector in this method can be 2Hz-10Hz.

[0155] Example 10: Investigation of CAD detector filter constants

[0156] S1. Solution preparation:

[0157] The preparation methods of mobile phase and reference solution are the same as those in Example 1.

[0158] S2. Chromatographic detection:

[0159] The CAD detector filter constant was set to 2s and 5s, and chromatographic column 1 was used for detection to investigate the effects of different detector filter constants on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as in Example 1.

[0160] S3. Chromatogram analysis:

[0161] The chromatograms corresponding to the CAD detector filter constants of 2s and 5s are as follows: Fig.23 , 24 .

[0162] according to Fig.23 As shown, when the filtering constant is 2s, the chromatographic peak with a retention time of 5.5min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.3min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.9min is the HEPES peak; according to Fig.24 When the filtering constant is 5s, the chromatographic peak with a retention time of 5.5min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.3min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.9min is the HEPES peak.

[0163] Therefore, the filter constant of the CAD detector in this method can be 2.0s-10.0s.

[0164] Example 11 Investigation of injection volume

[0165] S1. Solution preparation:

[0166] The preparation methods of mobile phase and reference solution are the same as those in Example 1.

[0167] S2. Chromatographic detection:

[0168] The injection volume was set to 10 μL and 50 μL, and the chromatographic column 1 was used for detection to investigate the effects of different injection volumes on the separation ability and retention time of sodium chloride and HEPES. Other chromatographic conditions were the same as in Example 1.

[0169] S3. Chromatogram analysis:

[0170] The corresponding chromatograms when the injection volume is 10μL and 50μL are as follows Fig.25 , 26 .

[0171] according to Fig.25As shown, when the injection volume is 10 μL, the chromatographic peak with a retention time of 5.6 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.3 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.9 min is the HEPES peak; according to Fig.26 As shown, when the injection volume is 50 μL, the chromatographic peak with a retention time of 5.3 min is the chloride ion peak of sodium chloride, the chromatographic peak with a retention time of 8.4 min is the sodium ion peak of sodium chloride, and the chromatographic peak with a retention time of 10.8 min is the HEPES peak.

[0172] Therefore, the injection volume in this method can be 10 μL-50 μL.

[0173] Example 12 Methodological Investigation

[0174] 12.1 Limit of Quantitation

[0175] Preparation of quantitative limit solution: 32.21 mg of sodium chloride and 16.10 mg of HEPES were accurately weighed and placed in a 50 mL volumetric flask, and a 90% methanol aqueous solution was added and ultrasonicated until completely dissolved and diluted to the scale as a reference substance stock solution. The reference substance stock solution was diluted in a series with a 90% methanol aqueous solution by volume, and the chromatographic conditions of Example 1 and chromatographic column 1 were used for investigation until the signal-to-noise ratio of the chloride ion peak and the HEPES peak in the chromatogram was about 10, and the quantitative limit solution was obtained.

[0176] The quantitative limit solution was sampled for 6 consecutive injections, and the quantitative limit concentrations of sodium chloride and HEPES were obtained according to the chromatographic conditions of Example 1 and detected using chromatographic column 1. The results are shown in Table 1.

[0177] Table 1 Quantitation limit results

[0178] sample Signal-to-Noise Ratio (S / N) Limit of Quantitation Percentage concentration Sodium chloride (chloride ion) 10 1.611μg / mL 0.96％ HEPES 9 4.025 μg / mL 4.97％

[0179] According to Table 1, the quantitative limits of sodium chloride and HEPES are 1.611 μg / mL and 4.025 μg / mL, respectively.

[0180] 12.2 Recovery rate

[0181] Linear reference solution: Accurately weigh 32.21 mg of sodium chloride and 16.10 mg of HEPES into a 50 mL volumetric flask, add 90% methanol aqueous solution by volume, sonicate until completely dissolved and dilute to the mark as reference stock solution. Accurately measure different volumes of reference stock solution, add 90% methanol aqueous solution by volume to dilute and prepare 40%, 80%, 100%, 160% and 200% solutions of the test concentration (i.e., a mixed solution containing 163 μg sodium chloride and 81 μg HEPES per mL) as linear reference solution. Accurately measure each concentration of reference solution and inject it into the liquid chromatograph to record the chromatogram. Calculate the linear regression equation of each substance with the concentration of each substance in the reference solution as the horizontal coordinate and the corresponding peak area as the vertical coordinate.

[0182] Recovery rate stock solution: accurately weigh 35.21 mg of sodium chloride and 16.48 mg of HEPES into a 50 mL volumetric flask, add 90% methanol aqueous solution by volume, sonicate until completely dissolved and dilute to the scale

[0183] Low concentration solution (80% test concentration): accurately measure 0.25 mL of irinotecan hydrochloride liposome solution, add 1.875 mL of recovery rate stock solution into a 25 mL volumetric flask, add 90% methanol, sonicate until completely dissolved and dilute to the scale.

[0184] Medium concentration solution (100% test concentration): accurately measure 0.25 mL of irinotecan hydrochloride liposome solution, add 3.125 mL of recovery rate stock solution into a 25 mL volumetric flask, add 90% methanol, sonicate until completely dissolved and dilute to the scale.

[0185] High concentration solution (120% test concentration): accurately measure 0.25 mL of irinotecan hydrochloride liposome solution, add 4.375 mL of recovery rate stock solution into a 25 mL volumetric flask, add 90% methanol, sonicate until completely dissolved and dilute to the scale.

[0186] The linear reference solution and the above-mentioned recovery rate solutions of each concentration were injected once each, and the chromatographic conditions of Example 1 were followed and the chromatographic column 1 was used for detection. The recovery rates of sodium chloride and HEPES were calculated. The results are shown in Table 2.

[0187] Table 2 Recovery results

[0188]

[0189] The results showed that the recovery rate of sodium chloride was between 101.1% and 101.4%, and the recovery rate of HEPES was between 99.5% and 104.0%, indicating that the method of the present application had good accuracy.

[0190] 12.3 Investigation of linear relationship

[0191] Take appropriate amounts of sodium chloride and HEPES, use 90% by volume methanol aqueous solution to ultrasonically dissolve the sample, and prepare a linear mixed solution containing 0.09 mg-0.37 mg of sodium chloride and 0.045 mg-0.18 mg of HEPES per mL.

[0192] Measure 20 μL of each of the above solutions and inject them into the chromatograph. According to the chromatographic conditions of Example 1, use chromatographic column 1 for detection and record the chromatogram. The results are shown in the attached Figure 27-28 .

[0193] according to Figure 27-28 It can be seen that the linear relationship is good in the range of 0.09mg / mL-0.37mg / mL for sodium chloride and in the range of 0.045mg / mL-0.18mg / mL for HEPES.

[0194] Example 13 Detection of the Content of Sodium Chloride and HEPES in Irinotecan Liposome Injection

[0195] Reference solution: Accurately weigh about 34 mg of sodium chloride and about 16 mg of HEPES into a 50 mL volumetric flask, add 90% methanol and sonicate until completely dissolved, dilute to the scale, and shake well; Accurately measure 5 mL of the solution into a 20 mL volumetric flask, dilute to the scale with 90% methanol, and shake well.

[0196] Test solution: accurately measure 1 mL of the reference preparation of irinotecan hydrochloride liposome injection and the homemade sample into a 50 mL volumetric flask, sonicate with 90% methanol until the liposomes are dissolved and diluted to the scale, shake well, and prepare two batches of each test sample; the reference preparation here is the commercially available irinotecan hydrochloride liposome injection, in which the concentrations of sodium chloride and HEPES are known.

[0197] 20 μL of each of the above solutions was measured and injected into the chromatograph, and the chromatographic conditions of Example 1 were followed, and the chromatographic column 1 was used for detection, and the chromatogram was recorded. The content of sodium chloride and HEPES in the reference preparation and the homemade sample was calculated by the external standard method. The calculation formula for the content of sodium chloride and HEPES in the test sample is as follows:

[0198]

[0199] Test results: The test spectra of the reference preparation of irinotecan liposome injection and the homemade sample are as follows: Figure 29-32 As shown in Table 3, the contents of sodium chloride and HEPES in the reference preparation and homemade samples were calculated according to the external standard method.

[0200] Table 3 Contents of sodium chloride and HEPES in reference preparations and homemade samples

[0201] sample Sodium chloride HEPES Reference preparation (20220815) 8.752mg / mL 4.281mg / mL Reference preparation (20220915) 8.637mg / mL 4.172mg / mL Homemade Sample(220805) 8.699mg / mL 4.084mg / mL Homemade Samples(220815) 8.843mg / mL 4.077mg / mL

[0202] The experimental results show that the sodium chloride and HEPES contents detected by this method fluctuate within the normal range of theoretical values, and there is no significant difference in the results of the reference preparation tested at different times.

[0203] The present application describes multiple embodiments, but the description is exemplary rather than restrictive, and it is obvious to a person skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application.

Claims

1. A method for simultaneously detecting multiple excipients in a preparation, wherein the excipients are sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid, and high performance liquid chromatography is used for detection, the method comprising the following steps: S1. Solution preparation: Prepare mobile phase; Prepare reference solution; Prepare test sample solution; S2. Detection: Injecting the reference solution and the test solution into a high performance liquid chromatograph for detection, and recording a chromatogram; S3. Analysis: According to the chromatograms of the reference solution and the test solution, the contents of sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid in the test sample were calculated by the external standard method; Among them, in the high performance liquid chromatography detection, the mobile phase consists of mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of volatile salt, and the mobile phase B is an organic solvent; the volume ratio of the mobile phase A to the mobile phase B is (25:75)-(20:80).

2. The method according to claim 1, wherein the preparation is a liposome injection, preferably, irinotecan hydrochloride liposome injection.

3. The method according to claim 1 or 2, wherein the mobile phase A is an aqueous solution of ammonium formate or ammonium acetate; and the mobile phase B is methanol or acetonitrile, preferably acetonitrile.

4. The method according to claim 3, wherein the volume ratio of mobile phase A to mobile phase B is 25:75, 24:76, 23:77, 22:78, 21:79 or 20:80; and / or The concentration of the mobile phase A is 50 mM-75 mM; preferably, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM or 75 mM; and / or The pH value of the mobile phase A is 2.5-3.5; preferably, 2.5-3.0 or 3.0-3.5; more preferably, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5; wherein, The pH adjuster of the mobile phase A is formic acid or acetic acid.

5. The method according to claim 4, in the high performance liquid chromatography detection, other chromatographic conditions include: Chromatographic column: bonded hydrophilic column; Chromatographic column model: Merck SeQuant ZIC-chilic (100×4.6mm, 3μm) chromatographic column, or Pheromones ComixslHCS (150×3.0mm, 3μm) chromatographic column; and / or Column temperature: 25°C-40°C, preferably, 25°C-30°C, 30°C-35°C or 35°C-40°C, more preferably, 25°C, 30°C, 35°C or 40°C; and / or Flow rate: 0.8 mL / min-1.2 mL / min, preferably, 0.8 mL / min-1.0 mL / min or 1.0 mL / min-1.2 mL / min, more preferably, 0.8 mL / min, 1.0 mL / min or 1.2 mL / min; and / or Elution mode: isocratic elution; Injection volume: 10 μL-50 μL, preferably 20 μL; Detector: Charged aerosol detector.

6. The method according to claim 5, wherein the condition of the detector is: Atomization temperature: 35°C or 55°C; and / or Data acquisition frequency: 2Hz-10Hz, preferably, 2Hz-5Hz or 5Hz-10Hz, more preferably, 2Hz, 3Hz, 4Hz, 5Hz, 6Hz, 7Hz, 8Hz, 9Hz or 10Hz; and / or Filter constant: 2.0s-10.0s, preferably, 2s-5s or 5s-10s, more preferably, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s or 10s.

7. The method according to claim 1 or 2, wherein in step S1, the reference solution is prepared by: dissolving sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid standard substance and a solubilizing agent in the same volumetric flask to prepare a reference stock solution; and diluting the reference stock solution to obtain the reference solution.

8. The method according to claim 7, wherein the concentrations of sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid in the reference solution are: 1.611 μg / ml-0.74 mg / ml sodium chloride, 4.025 μg / ml-0.36 mg / ml 4-hydroxyethylpiperazineethanesulfonic acid; preferably, 163 μg / ml sodium chloride and 81 μg / ml 4-hydroxyethylpiperazineethanesulfonic acid.

9. The method according to claim 1 or 2, wherein in step S1, the test solution is prepared by dissolving a test sample containing sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid in a solubilizing agent to prepare a test solution.

10. The method according to claim 1 or 2, wherein in step S1, the solvent of the reference solution and the test solution is a 90% by volume methanol aqueous solution.

11. The method according to claim 1 or 2, in step S3, the calculation formula for the content of sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid in the test sample is as follows:

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