Liquid chromatography separation and detection method for DODMA and oxidative degradation impurities thereof and application of liquid chromatography separation and detection method

Abstract

The invention discloses a liquid chromatography separation and detection method and application of DODMA and oxidative degradation impurities thereof. The detection method comprises the following steps: preparing DODMA oxidative degradation impurity solutions with different concentrations as standard solutions, and preparing test solutions containing the oxidative degradation impurities; a silica gel column with octadecyl or octyl is used as a stationary phase, an acetic acid-water-ammonia water solution is used as a mobile phase A, methanol is used as a mobile phase B, and the standard solution and the test solution are subjected to gradient elution; and after the elution is finished, carrying out evaporative light detection on the eluent, establishing a standard curve of Lg concentration-Lg peak area according to the detection signal intensity of the eluent of the standard solution, and then calculating according to the signal intensity of the test solution and a linear external standard method to obtain the content of the oxidative degradation impurities in the test solution. The method can well separate DODMA and oxidative degradation impurities thereof, and is simple to operate, high in detection efficiency, and good in reproducibility and stability.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical detection, and in particular relates to a liquid chromatography separation and detection method for DODMA and its oxidative degradation impurities and an application thereof. Background Art

[0002] 1,2-Dioleyl alcohol-3-dimethylamino-propane (DODMA) is a fully synthetic ionizable cationic lipid and a good membrane material for cationic liposomes. It is a lipophilic nonionic surfactant and a compound naturally present in some plants.

[0003] DODMA can be obtained by reducing oleic acid with lithium aluminum hydride to produce oleyl alcohol, which is then esterified with methylsulfonyl chloride and condensed with 3-dimethylamino-1,2-propanediol under alkaline conditions to form an ether. During the oxidative destruction process, it was found that DODMA (Formula 1) would degrade under oxidative conditions to generate an oxidative degradation impurity (Formula 2). This impurity is difficult to detect and separate in DODMA, which greatly affects the quality control and application safety of DODMA products.

[0004]

[0005] Therefore, in view of the above technical problems, it is necessary to provide a liquid chromatography separation and detection method and application of DODMA and its oxidative degradation impurities.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] The object of the present invention is to provide a liquid chromatography separation and detection method for DODMA and its oxidative degradation impurities and application.

[0008] In order to achieve the above object, a technical solution provided by a specific embodiment of the present invention is as follows:

[0009] The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities comprises:

[0010] The DODMA oxidative degradation impurity solutions of different concentrations are prepared as standard solutions and test solutions containing oxidative degradation impurities, specifically, by accurately weighing an appropriate amount of oxidative degradation impurity reference substance, adding methanol to dissolve it, and preparing standard curve solutions of different concentrations of 2 to 20 μg / ml; accurately weighing an appropriate amount of DODMA, adding methanol to dissolve it, and preparing a 2 mg / ml solution to obtain the test solution;

[0011] Using octadecyl or octyl silica gel column as stationary phase, acetic acid-water-ammonia solution as mobile phase A, methanol solution as mobile phase B, the standard solution and the test solution are gradient eluted;

[0012] After elution, the eluate was detected by an evaporative light detector, and a standard curve of Lg concentration-Lg peak area was established based on the detection signal intensity of the eluate of the standard solution. Then, based on the signal intensity of the test solution, the content of oxidative degradation impurities in the test solution was calculated according to the linear external standard method.

[0013] In one or more embodiments of the present invention, mobile phase A is an aqueous solution of acetic acid with a volume ratio of 0.1% to 0.5%, and the pH is adjusted to 5.8 to 6.2 with aqueous ammonia.

[0014] In one or more embodiments of the present invention, mobile phase B is 80% to 100% methanol aqueous solution.

[0015] In one or more embodiments of the present invention, the procedure of gradient elution is:

[0016] From 0 min to 10 min, the state of the mobile phase changes from the starting point to the first transition point, the starting point satisfies the volume ratio of mobile phase A: mobile phase B is 20:80, and the first transition point satisfies the volume ratio of mobile phase A: mobile phase B is 0:100;

[0017] Maintain the first transition point state for 40 minutes;

[0018] From 40min to 41min, the state of the mobile phase changes from the first transition point to the second transition point, where the volume ratio of mobile phase A:mobile phase B is 20:80, and the second transition point state is maintained until 45min.

[0019] In one or more embodiments of the present invention, the stationary phase has the following specifications during gradient elution: inner diameter 4.6-6.0 mm, length 150-250 mm, filler particle size 3-5 μm. Preferably, the chromatographic column has the following specifications: inner diameter 4.6 mm, length 150 mm, filler particle size 3.5 μm.

[0020] In one or more embodiments of the present invention, the column temperature during gradient elution is 25° C. to 40° C. Preferably, the column temperature is 30° C.

[0021] In one or more embodiments of the present invention, the mobile phase flow rate during gradient elution is 0.8-1.2 ml / min. Preferably, the mobile phase flow rate is 1.0 ml / min.

[0022] In one or more embodiments of the present invention, the injection volume during gradient elution is 20-50 μl. Preferably, the injection volume is 30 μl.

[0023] In one or more embodiments of the present invention, the temperature of the evaporation tube during evaporation light detection is 55-65° C. Preferably, the temperature of the evaporation tube is 60° C.

[0024] In one or more embodiments of the present invention, the temperature of the atomization tube during evaporative light detection is 45-60° C. Preferably, the temperature of the atomization tube is 55° C.

[0025] In one or more embodiments of the present invention, the gas flow rate during evaporative light detection is 1.0-2.8 SLM. Preferably, the gas flow rate is 1.6 SLM.

[0026] In one or more embodiments of the present invention, a liquid chromatography separation and detection method for DODMA and its oxidative degradation impurities is used in the detection of DODMA products.

[0027] Compared with the prior art, the liquid chromatography separation and detection method for DODMA and its oxidative degradation impurities and its application of the present invention can well separate DODMA and its oxidative degradation impurities, and has simple operation, high detection efficiency, and good method reproducibility and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a graph showing the specificity of a preferred method in one embodiment of the present invention;

[0030] Figure 2 is a logarithmic linear relationship diagram of the logarithm of the peak area and the concentration in one embodiment of the present invention;

[0031] Figure 3 This is a specific standard curve diagram of a flow rate of 0.8 ml / min in one embodiment of the present invention;

[0032] Figure 4 This is a specific standard curve diagram of a flow rate of 1.2 ml / min in one embodiment of the present invention;

[0033] Figure 5 This is a pH 5.8 specific standard curve diagram in one embodiment of the present invention;

[0034] Figure 6 This is a pH 6.2-specific standard curve diagram in one embodiment of the present invention;

[0035] Figure 7This is a standard curve diagram specific to 65°C temperature of the evaporation tube in one embodiment of the present invention;

[0036] Figure 8 This is a standard curve diagram specific to 55°C evaporation tube temperature in one embodiment of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0038] The present invention provides a liquid chromatography separation and detection method for DODMA and its oxidative degradation impurities, which may include the following steps:

[0039] (1) preparing oxidative degradation impurity solutions of different concentrations and DODMA test solutions containing oxidative degradation impurities;

[0040] (2) using a silica gel column with octadecyl or octyl as the stationary phase, acetic acid-water-ammonia solution as the mobile phase A, and methanol solution as the mobile phase B, to perform gradient elution on the oxidative degradation impurity solution and the test solution;

[0041] (3) After elution, an evaporative light detector (ELSD) is used to detect the signal intensity of the test solution, and the content of oxidative degradation impurities in the test solution is calculated according to the linear external standard method.

[0042] In a specific embodiment, the specific operation of step (1) is: accurately weigh an appropriate amount of oxidative degradation impurity reference substance, add methanol to dissolve it, and prepare a standard curve solution of different concentrations of 2 to 20 μg / ml; accurately weigh an appropriate amount of DODMA, add methanol to dissolve it, and prepare a 2 mg / ml solution to obtain the test solution.

[0043] In a specific embodiment, the specific operation of step (1) is as follows: accurately weigh an appropriate amount of oxidative degradation impurity reference substance, dissolve it in methanol, and prepare standard curve solutions with concentrations of 1.0 μg / ml, 4.0 μg / ml, 8.0 μg / ml, and 20.0 μg / ml. Accurately weigh an appropriate amount of DODMA, dissolve it in methanol, and prepare a 2 mg / ml solution.

[0044] In some specific embodiments, step (2) is specifically as follows: using a silica gel column with octadecyl as the stationary phase, the specifications of the chromatographic column are: inner diameter 4.6-6.0 mm, length 150-250 mm, filler particle size 3-5 μm, mobile phase A is acetic acid-water with a volume ratio of 1-5:1000, adjusted to pH 5.8-6.2 with ammonia water, mobile phase B is 80%-100% methanol aqueous solution, column temperature is 25°C-40°C, and mobile phase flow rate is 0.8-1.2 ml / m in, the injection volume was 20-50 μl, and the standard curve solution and the test solution were gradient eluted to separate DODMA and its oxidative degradation impurities. The gradient elution program was: from 0 min to 10 min, the mobile phase A: mobile phase B-20:80 changed to mobile phase A: mobile phase B-0:100, maintained to 40 min, from 40 min to 41 min, the mobile phase A: mobile phase B-0:100 changed to mobile phase A: mobile phase B-20:80, maintained to 45 min.

[0045] In a specific embodiment, step (2) is specifically as follows: using a silica gel column with octadecyl as the stationary phase, the chromatographic column model is Waters Xbridge Shield RP18, the specifications of the chromatographic column are: inner diameter 4.6mm, length 150mm, filler particle size 3.5μm, mobile phase A is acetic acid-water with a volume ratio of 2:1000, adjusted to pH 6.0 with ammonia water, mobile phase B is methanol solution, column temperature is 30°C, mobile phase flow rate is 1.0ml / min, injection volume is 30μl, gradient elution is performed on the standard curve solution and the test solution to separate DODMA and its oxidative degradation impurities, the gradient elution is changed from mobile phase A: mobile phase B-20:80 to mobile phase A: mobile phase B-0:100 from 0min to 10min, maintained to 40min, and from 40min to 41min, changed from mobile phase A: mobile phase B-0:100 to mobile phase A: mobile phase B-20:80, maintained to 45min.

[0046] In some specific embodiments, the temperature of the ELSD evaporation tube in step (3) is 55-65°C; the temperature of the atomization tube is 45-60°C; and the gas flow rate is 1.0-2.8 SLM.

[0047] In a specific embodiment, the temperature of the ELSD evaporation tube in step (3) is 60° C.; the temperature of the atomization tube is 55° C.; and the gas flow rate is 1.6 SLM.

[0048] If no specific experimental steps or conditions are specified in the following examples, the experiments can be carried out according to the conventional experimental steps or conditions in the art.

[0049] The instruments, reagents and samples used in the present invention are all conventional products that can be purchased commercially, including but not limited to the instruments, reagents and samples shown below.

[0050] Table 1 Instruments and reagents used in the embodiments of the present invention

[0051] name Model / Specification Manufacturer High performance liquid chromatography Agilent1260 Agilent Electronic balance XS105DU / 1 / 100,000 Sartorius Electronic balance XPR2 / A / 1 / million Mettler Water Purifier Milli-Q Millipore Methanol (MeOH) HPLC grade / 4L / bottle Adamas Acetic acid AR grade / 500mL / bottle Sinopharm Ultrapure water HPLC grade Homemade water purifier ammonia AR-level Sinopharm

[0052] Table 2 Samples used in the embodiments of the present invention

[0053] name content(%) Manufacturer Oxidative degradation of impurities 91.9％ Suzhou Seaview Pharmaceutical Technology Co., Ltd. DODMA 97.6％ Tianfang Pharmaceutical Co., Ltd.

[0054] Embodiment group 1:

[0055] Example 1: Specificity test

[0056] Solvent: Methanol

[0057] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0058] Standard curve solution:

[0059] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0060] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0061] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0062] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0063] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0064] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0065] Table 3 Chromatographic conditions for specificity test

[0066]

[0067]

[0068] System suitability requirements: The standard curve uses Lg concentration as the horizontal axis (X) and Lg peak area as the vertical axis (Y). Linear regression analysis is performed and the linear regression coefficient (r) should not be less than 0.990. Figure 1 shown.

[0069] In the spiked test sample, the oxidative degradation impurity detection result was 0.21%.

[0070] Example 2: Sensitivity test

[0071] Quantitation limit solution: Accurately measure 3 ml of the standard curve LIN3 solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0072] Detection limit solution: Accurately measure 8 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0073] Accurately measure 30 μl of the quantitative limit solution and the detection limit solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 4.

[0074] Table 4 Sensitivity test results

[0075]

[0076] From the results in Table 4, it can be seen that the quantitative limit sensitivity of the detection method of the present invention is 1.100 μg / ml; the detection limit sensitivity is 0.8800 μg / ml, and the sensitivity meets the requirements.

[0077] Example 3: Linearity and Range

[0078] Take an appropriate amount of oxidative degradation impurities, dilute with solvent to prepare a series of concentrations of reference solution, inject into liquid chromatograph, record the chromatogram, and the results are shown in Table 5.

[0079] Table 5 Oxidative degradation impurity linearity

[0080]

[0081] The results show that if Figure 2 As shown, within the concentration range of 1.100 μg / ml to 18.34 μg / ml for oxidative degradation impurities, the logarithm of the peak area has a good linear relationship with the logarithm of the concentration, and the correlation coefficient is greater than 0.999.

[0082] Example 4: Accuracy

[0083] Spiked test solution: Take 20 mg of DODMA, weigh accurately, place in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml. Prepare 6 copies in parallel.

[0084] Accurately measure 30 μl of each of the above-mentioned spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 6.

[0085] Table 6 Accuracy results

[0086]

[0087] The results showed that the accuracy and repeatability were good, the test results of 6 spiked test solutions met the requirements, the recoveries were 86.6% to 92.1%, and the RSD of the 6 test results was 2.7%.

[0088] The present invention relates to a liquid chromatography separation and detection method for DODMA and its oxidative degradation impurities, comprising the following steps: (1) preparing oxidative degradation impurity solutions of different concentrations and a DODMA test solution containing oxidative degradation impurities; (2) using a silica gel column with octadecyl as the stationary phase, acetic acid-water-ammonia water as the mobile phase A, and a methanol solution as the mobile phase B, and gradient eluting the DODMA test solution containing oxidative degradation impurities through a high performance liquid chromatography system; (3) entering an evaporative light detector after the elution, in which the solvent is removed in a gaseous form, leaving a sample, and then detecting the scattered light of the sample through a light scattering detector to obtain the signal intensity of the sample to be tested, and calculating the content of the oxidative degradation impurities in the test solution according to the external standard method. The detection method of the present invention can well detect oxidative degradation impurities, is simple to operate, has a fast peak time, high detection efficiency, and good method reproducibility and stability.

[0089] Embodiment Group 2

[0090] Example 11: Specificity test at flow rate 0.8 ml / min

[0091] Solvent: Methanol

[0092] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0093] Standard curve solution:

[0094] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0095] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0096] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0097] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0098] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0099] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0100] Table 7 Chromatographic conditions for specificity test

[0101]

[0102]

[0103] System suitability requirements: The standard curve uses Lg concentration as the horizontal axis (X) and Lg peak area as the vertical axis (Y). Linear regression analysis is performed and the linear regression coefficient (r) should not be less than 0.990. Figure 3 shown.

[0104] In the spiked test sample, oxidative degradation impurities were detected at 0.20%.

[0105] Example 12: Specificity test at flow rate 1.2 ml / min

[0106] Solvent: Methanol

[0107] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0108] Standard curve solution:

[0109] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0110] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0111] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0112] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0113] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0114] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0115] Table 8 Chromatographic conditions for specificity test

[0116]

[0117] System suitability requirements: The standard curve uses Lg concentration as the horizontal axis (X) and Lg peak area as the vertical axis (Y). Linear regression analysis is performed and the linear regression coefficient (r) should not be less than 0.990. Figure 4 shown.

[0118] In the spiked test sample, oxidative degradation impurities were detected at 0.21%.

[0119] Comparative Example 1: Specificity test at flow rate 1.5 ml / min

[0120] Solvent: Methanol

[0121] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0122] Standard curve solution:

[0123] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0124] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0125] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0126] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0127] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0128] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0129] Table 9 Chromatographic conditions for specificity test

[0130]

[0131]

[0132] Oxidative degradation impurities were not separated from the remaining impurities in DODMA.

[0133] Embodiment Group 3

[0134] Example 21: pH 5.8 specificity test

[0135] Solvent: Methanol

[0136] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0137] Standard curve solution:

[0138] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0139] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0140] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0141] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0142] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0143] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0144] Table 10 Chromatographic conditions for specificity test

[0145]

[0146] System suitability requirements: The standard curve uses Lg concentration as the horizontal axis (X) and Lg peak area as the vertical axis (Y). Linear regression analysis is performed and the linear regression coefficient (r) should not be less than 0.990. Figure 5 shown.

[0147] In the spiked test sample, oxidative degradation impurities were detected at 0.22%.

[0148] Example 22: pH 6.2 specificity test

[0149] Solvent: Methanol

[0150] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0151] Standard curve solution:

[0152] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0153] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0154] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0155] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0156] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0157] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0158] Table 11 Chromatographic conditions for specificity test

[0159]

[0160] System suitability requirements: The standard curve uses Lg concentration as the horizontal axis (X) and Lg peak area as the vertical axis (Y). Linear regression analysis is performed and the linear regression coefficient (r) should not be less than 0.990. Figure 6 shown.

[0161] In the spiked test sample, oxidative degradation impurities were detected at 0.21%.

[0162] Comparative Example 2: pH 5.5 specificity test

[0163] Solvent: Methanol

[0164] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0165] Standard curve solution:

[0166] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0167] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0168] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0169] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0170] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0171] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0172] Table 12 Chromatographic conditions for specificity test

[0173]

[0174]

[0175] Oxidative degradation impurities were not separated from the remaining impurities in DODMA.

[0176] Embodiment Group 4

[0177] Example 31: Specificity test at 65°C evaporation tube temperature

[0178] Solvent: Methanol

[0179] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0180] Standard curve solution:

[0181] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0182] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0183] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0184] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0185] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0186] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0187] Table 13 Chromatographic conditions for specificity test

[0188]

[0189] System suitability requirements: The standard curve uses Lg concentration as the horizontal axis (X) and Lg peak area as the vertical axis (Y). Linear regression analysis is performed and the linear regression coefficient (r) should not be less than 0.990. Figure 7 shown.

[0190] In the spiked test sample, oxidative degradation impurities were detected at 0.19%.

[0191] Example 32: Specificity test at 55°C evaporation tube temperature

[0192] Solvent: Methanol

[0193] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0194] Standard curve solution:

[0195] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0196] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0197] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0198] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0199] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0200] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0201] Table 14 Chromatographic conditions for specificity test

[0202]

[0203]

[0204] System suitability requirements: The standard curve uses Lg concentration as the horizontal axis (X) and Lg peak area as the vertical axis (Y). Linear regression analysis is performed and the linear regression coefficient (r) should not be less than 0.990. Figure 8 shown.

[0205] In the spiked test sample, oxidative degradation impurities were detected at 0.20%.

[0206] Comparative Example 3: Specificity test at 70°C evaporation tube temperature

[0207] Solvent: Methanol

[0208] Stock solution of oxidative degradation impurity reference substance: Accurately weigh an appropriate amount of oxidative degradation impurity reference substance, equivalent to 4 mg of oxidative degradation impurities, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0209] Standard curve solution:

[0210] LIN1-20μg / ml: Accurately measure 2ml of the oxidative degradation impurity reference stock solution, place it in a 20ml volumetric flask, dilute to the scale with solvent, and shake well.

[0211] LIN2-8μg / ml: Accurately measure 4ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0212] LIN3-4μg / ml: Accurately measure 2ml of LIN1 solution, place in a 10ml volumetric flask, dilute to the mark with solvent, and shake well.

[0213] LIN4-2μg / ml: Accurately measure 1ml of LIN1 solution, place it in a 10ml volumetric flask, dilute to the scale with solvent, and shake well.

[0214] DODMA test solution: Take 20 mg of DODMA, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with solvent, and shake well.

[0215] Spiked test solution: Take 20 mg of DODMA, accurately weigh it, put it in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 2 ml of standard curve LIN1 solution, dilute to the scale with solvent, shake well, and prepare a solution containing 2 mg of DODMA and 0.004 mg of oxidative degradation impurities per 1 ml.

[0216] Table 15 Chromatographic conditions for specificity test

[0217]

[0218] Oxidative degradation impurities have low response to DODMA.

[0219] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

[0220] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0221] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A liquid chromatography separation and detection method for DODMA and its oxidative degradation impurities, characterized in that: include: preparing DODMA oxidative degradation impurity solutions of different concentrations as standard solutions and test solutions containing oxidative degradation impurities; Using a silica gel column with octadecyl or octyl as the stationary phase, acetic acid-water-ammonia solution as the mobile phase A, and methanol solution as the mobile phase B, the standard solution and the test solution are subjected to gradient elution; After elution, the eluate was subjected to evaporative light detection, and a standard curve of Lg concentration-Lg peak area was established based on the detection signal intensity of the eluate of the standard solution. Then, based on the signal intensity of the test solution, the content of oxidative degradation impurities in the test solution was calculated according to the linear external standard method.

2. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The mobile phase A is an aqueous solution prepared by mixing acetic acid and water in a volume ratio of 1 to 5:1000, and then adjusted to a pH of 5.8 to 6.2 with aqueous ammonia.

3. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The mobile phase B is 80% to 100% methanol aqueous solution.

4. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The procedure of the gradient elution is: From 0 min to 10 min, the state of the mobile phase changes from the starting point to the first transition point, the starting point satisfies the volume ratio of mobile phase A: mobile phase B is 20:80, and the first transition point satisfies the volume ratio of mobile phase A: mobile phase B is 0:100; Maintain the first transition point state for 40 minutes; From 40min to 41min, the state of the mobile phase changes from the first transition point to the second transition point, where the volume ratio of mobile phase A:mobile phase B is 20:80, and the second transition point state is maintained until 45min.

5. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The column temperature during the gradient elution is 25°C to 40°C.

6. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The flow rate of the mobile phase during the gradient elution is 0.8-1.2 ml / min.

7. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The injection volume during the gradient elution is 20-50 μl.

8. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The temperature of the evaporation tube during the evaporation light detection is 55-65°C.

9. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The temperature of the atomizing tube during the evaporative light detection is 45-60°C.

10. The liquid chromatography separation and detection method of DODMA and its oxidative degradation impurities according to claim 1, characterized in that: The gas flow rate during the evaporative light detection is 1.0-2.8 SLM.

11. Use of the liquid chromatography separation and detection method for DODMA and its oxidative degradation impurities according to any one of claims 1 to 10 in the detection of DODMA products.

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