Separation method of related substances of phospholipid-containing preparation

Abstract

The invention provides a method for separating related substances in a phospholipid-containing preparation, which adopts a high performance liquid chromatography, a chromatographic column adopts octadecylsilane chemically bonded silica as a filler, mobile phases comprise a mobile phase A, a mobile phase B and an optional mobile phase C, and a gradient elution procedure and an electrospray detector are adopted for separation and detection. The separation method provided by the invention is strong in specificity, good in separation degree, high in sensitivity, good in accuracy, simple and convenient to operate and capable of effectively and accurately monitoring the quality change condition of the phospholipid-containing preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for separating related substances of a phospholipid-containing preparation. Background Art

[0002] Preparations containing phospholipids have been widely studied in new drug preparations due to their safety and targeted positioning. Common phospholipids include but are not limited to lecithin, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, etc., as well as the above phospholipids' derivatives, palmitic acid derivatives, stearic acid derivatives, etc. During the preparation and storage of the preparation, phospholipids are degraded to produce related substances such as palmitic acid, stearic acid, polyethylene glycol, lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE). Among them, palmitic acid and stearic acid are easy to react with thickeners commonly used in preparations, such as propylene glycol and glycerol, to generate palmitoyl propanediol, palmitic acid glyceride, stearyl propanediol, and stearic acid glyceride. Quantitative detection of these related substances can reflect the quality of the preparation and effectively control the safety and effectiveness of the drug preparation.

[0003] Phospholipid-related substances such as LPA do not absorb ultraviolet light and cannot excite fluorescence, so ultraviolet detectors or fluorescence detectors cannot be used, which makes the development of separation methods very difficult. In the prior art, phospholipid substances and their related substances separation methods mostly use evaporative light detectors, such as CN115808483A and CN111253434A. However, evaporative light detectors have a low response to LPA and have limitations in use.

[0004] Therefore, developing a separation method for related substances with good separation effect and high sensitivity is of great significance for the quality control of phospholipid-containing preparations. Summary of the invention

[0005] The purpose of the present invention is to provide a method for separating related substances in a phospholipid-containing preparation, which can quantitatively detect the related substances and can be used to accurately monitor the quality changes of the preparation.

[0006] The invention provides a method for separating related substances in a phospholipid-containing preparation. The separation method adopts a high performance liquid chromatography method, a chromatographic column adopts octadecylsilane bonded silica gel as a filler, and a mobile phase comprises a mobile phase A, a mobile phase B and an optional mobile phase C.

[0007] In some embodiments, the related substances include one or more of LPA, palmitic acid, and palmitoyl propanediol.

[0008] In some embodiments, in the separation method provided by the present invention, the pore size of the chromatographic column is For example Best

[0009] In some embodiments, in the separation method provided by the present invention, the mobile phase is mobile phase A, mobile phase B and mobile phase C.

[0010] In some embodiments, in the separation method provided by the present invention, the mobile phase is mobile phase A and mobile phase B.

[0011] In some embodiments, in the separation method provided by the present invention, the mobile phase A is a buffered saline solution, and the mobile phase B and the mobile phase C are both organic solvents.

[0012] In some embodiments, in the separation method provided by the present invention, the organic solvent is selected from one or more of methanol, acetonitrile, tetrahydrofuran, ethanol, acetone, n-propanol, isopropanol and N,N-dimethylformamide.

[0013] In some embodiments, in the separation method provided by the present invention, the mobile phase B is acetonitrile.

[0014] In some embodiments, in the separation method provided by the present invention, the mobile phase C is methanol.

[0015] In some embodiments, in the separation method provided by the present invention, the mobile phase A is a volatile buffered saline solution.

[0016] In some embodiments, in the separation method provided by the present invention, the mobile phase A is selected from one or both of ammonium formate buffered saline solution and ammonium acetate buffered saline solution, preferably ammonium formate buffered saline solution.

[0017] In some embodiments, in the separation method provided by the present invention, the buffer salt concentration in the mobile phase A is 10-50 mM, preferably 20-40 mM, more preferably 20-30 mM, more preferably 24-26 mM, and more preferably 25 mM.

[0018] In some embodiments, in the separation method provided by the present invention, the pH of the mobile phase A is 2.5 to 3.5, preferably 2.8 to 3.2, and more preferably 3.0. In some embodiments, in the separation method provided by the present invention, the elution procedure of the mobile phase is: 0-3min: 50% mobile phase A, 50% mobile phase B, 0% mobile phase C; 3-20min: mobile phase A gradually decreases to 40%, mobile phase B gradually increases to 60%, 0% mobile phase C; 20-25min: mobile phase A gradually decreases to 20%, mobile phase B gradually increases to 80%, 0% mobile phase C; 25-35min: mobile phase A gradually decreases to 10%, mobile phase B gradually increases to 90%, 0% mobile phase C; 35-40min: mobile phase A gradually decreases to 0%, mobile phase B gradually decreases to 0%, mobile phase C gradually increases to 100%; 40-50min: mobile phases A, B, and C all remain unchanged; 50-55min: mobile phase A gradually increases to 50%, mobile phase B gradually increases to 50%, and mobile phase C gradually decreases to 0%; 55-60min: mobile phases A, B, and C all remain unchanged.

[0019] In some embodiments, in the separation method provided by the present invention, the elution procedure of the mobile phase is: 0min: 60% mobile phase A, 40% mobile phase B; 0-35min: mobile phase A gradually decreases to 10%, mobile phase B gradually increases to 90%; 35-45min: mobile phases A and B remain unchanged; 45-50min: mobile phase A gradually increases to 60%, mobile phase B gradually decreases to 40%; 50-60min: mobile phases A and B remain unchanged.

[0020] In some embodiments, in the separation method provided by the present invention, the flow rate of the mobile phase is 1.0-1.5 ml / min, for example, 1.0 ml / min, 1.1 ml / min, 1.2 ml / min, 1.3 ml / min, 1.4 ml / min, 1.5 ml / min, preferably 1.0-1.3 ml / min, preferably 1.0-1.2 ml / min, preferably 1.1-1.3 ml / min, and further preferably 1.2 ml / min.

[0021] In some embodiments, in the separation method provided by the present invention, the temperature of the chromatographic column is 30-40°C, for example, 30°C, 33°C, 35°C, 37°C, 40°C, preferably 33-37°C, and more preferably 35°C.

[0022] In some embodiments, in the separation method provided by the present invention, the injection volume used is 50-100 μl, for example, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 1000 μl, preferably 100 μl.

[0023] In some embodiments, in the separation method provided by the present invention, the detector used is an evaporative light scattering detector (ELSD) or an electrospray ionization detector (CAD), preferably CAD.

[0024] In some embodiments, in the separation method provided by the present invention, the nebulizer temperature of the CAD detector is 35-60°C, for example, 35°C, 40°C, 45°C, 48°C, 50°C, 52°C, 55°C, 60°C, preferably 40-60°C, more preferably 45-55°C, more preferably 48-52°C, and more preferably 50°C.

[0025] In some embodiments, the separation method provided by the present invention can be used for continuous detection and maintaining a stable baseline.

[0026] Beneficial Effects

[0027] The high performance liquid chromatography separation method provided by the present invention realizes accurate detection of related substances with low response intensity in phospholipid-containing preparations by screening specific mobile phases and elution gradients and other conditions. The method has strong specificity, good separation, high sensitivity, good accuracy, and a stable baseline for continuous injection of multiple samples, which can realize continuous detection of large batches of samples. The operation is simple and the samples do not need any pretreatment and can be directly analyzed on the machine.

[0028] The high performance liquid chromatography detection method provided by the present invention has a wide range of durability, fully complies with the guiding principles of methodological verification of the Chinese Pharmacopoeia in terms of system applicability and accuracy, can effectively and accurately reflect the quality changes of phospholipid-containing preparations, and strictly control the safety of drugs.

[0029] Definitions and terms

[0030] As described in the present invention, "palmitoyl propanediol" refers to the product of esterification of palmitic acid and 1,2-propylene glycol, and its CAS number is 3539-33-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Chromatogram obtained by the detection method of condition 1 of Example 2;

[0032] Figure 2 Chromatogram obtained by the detection method of condition 2 of Example 2;

[0033] Figure 3 Chromatogram obtained by the detection method of Example 3 with the atomizer temperature at 40°C;

[0034] Figure 4 Chromatogram obtained by the detection method of Example 3 with the atomizer temperature at 50°C;

[0035] Figure 5Chromatogram obtained by the detection method of Example 3 with the atomizer temperature at 60°C;

[0036] Figure 6 Chromatogram obtained by the detection method of Example 6;

[0037] Figure 7 Chromatogram obtained by the gradient elution program detection method of Comparative Example 1;

[0038] Figure 8 Chromatogram obtained by isocratic elution program detection method of comparative example 1;

[0039] Fig. 9 Chromatogram obtained by the detection method of Comparative Example 3. DETAILED DESCRIPTION

[0040] In order to make the technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0041] Example 1

[0042] Method for detecting related substances in octafluoropropane lipid microsphere injection by high performance liquid chromatography.

[0043] (1) Solution preparation

[0044] Test solution: octafluoropropane lipid microsphere injection.

[0045] Stock solutions of related substance reference substances: Weigh palmitic acid, palmitoyl propanediol, and LPA as related substance reference substances respectively, add methanol to dissolve them, and prepare stock solutions of related substance reference substances respectively.

[0046] Mix the stock solutions of related substance reference substances: Take an appropriate amount of the stock solutions of each related substance reference substance and dilute them into a methanol solution containing 30 μg / mL LPA, 50 μg / mL palmitic acid and 20 μg / mL palmitoyl propanediol.

[0047] System suitability solution: Accurately pipette 0.5 mL of the mixed related substance reference stock solution into a 2 mL volumetric flask, add 1 mL of the test solution, and dilute to the mark with methanol.

[0048] (2) Chromatographic conditions

[0049] Chromatographic column 1: Octadecylsilane bonded silica as filler, pore size

[0050] Mobile phase A: 25 mM ammonium formate buffered saline solution, pH 3.0; Mobile phase B: acetonitrile; Mobile phase C: methanol;

[0051] Flow rate: 1.2 ml / min;

[0052] The column temperature was 35°C; the injection volume was 100 μl;

[0053] Detector: CAD; Nebulizer temperature: 50°C

[0054] The gradient elution program is shown in Table 1.

[0055] Table 1 Gradient elution program

[0056]

[0057]

[0058] (3) Calculation method

[0059] The content of related substances was calculated by peak area using the external standard method. The calculation formula is:

[0060]

[0061] in:

[0062] A t It indicates the peak area of ​​the relevant substance in the chromatogram of the test solution;

[0063] V t Indicates the dilution multiple of the test solution, ml;

[0064] A s It indicates the peak area of ​​the relevant substance in the chromatogram of the reference solution;

[0065] V s Indicates the dilution multiple of the reference solution, ml;

[0066] W s Indicates the sample weight of the relevant substance reference substance, mg;

[0067] 1000 represents the mass conversion factor, 1000 μg / mg.

[0068] P indicates the purity of the reference substance.

[0069] (4) Test results

[0070] The separation of each component in the chromatogram of the system suitability solution is greater than 1.5, and the peak shape is symmetrical, indicating that the detection method of the present invention has good specificity and the responses of related substances of the detection method meet the requirements.

[0071] (5) Limit of quantification and limit of detection

[0072] The reference stock solutions of the relevant substances were diluted step by step with methanol, with a signal-to-noise ratio of about 3:1 as the detection limit and a signal-to-noise ratio of about 10:1 as the quantification limit, and the injection was repeated 6 times. The results are shown in Table 2. The detection method of the present invention has a stable baseline for continuous injection, the sensitivity meets the detection requirements, the precision is good, and accurate detection of large quantities of samples can be achieved.

[0073] Table 2 Results of investigation on limit of quantitation and limit of detection

[0074]

[0075] Example 2

[0076] Investigating the effect of column temperature on the detection method

[0077] (1) Solution preparation

[0078] Mixed related substance reference solution 1: Take appropriate amount of LPC, LPA, palmitic acid, and palmitoyl propanediol reference substances, add methanol to dissolve, and quantitatively dilute to make a mixed solution containing 10μg LPC, 7.5μg LPA, 12.5μg palmitic acid, and 5μg palmitoyl propanediol per 1ml, and shake well.

[0079] (2) Chromatographic conditions

[0080] Condition 1: flow rate: 1.0 ml / min, column temperature 30 °C;

[0081] Condition 2: flow rate: 1.0 ml / min, column temperature 35 °C;

[0082] The remaining chromatographic parameters are as follows:

[0083] Chromatographic column 2: Octadecylsilane bonded silica as filler, pore size

[0084] Mobile phase A: 25 mM ammonium formate buffered saline solution, pH 3.0; Mobile phase B: acetonitrile;

[0085] Injection volume: 100 μl;

[0086] Detector: CAD; Nebulizer temperature: 35°C

[0087] The gradient elution program is shown in Table 3.

[0088] Table 3 Gradient elution program

[0089] Time / minute 0 35 45 50 60 A％ 60 10 10 60 60 B％ 40 90 90 40 40

[0090] (3) Test results

[0091] The mixed related substance reference solution 1 was injected for testing. The detection methods of conditions 1 to 2 can accurately detect the related substances in the phospholipid-containing preparations. The chromatogram is as follows: Figure 1-2 As shown in the figure, LPC, LPA, palmitic acid and palmitoyl propanediol can be effectively detected. As the column temperature increases, the retention time of the relevant substances becomes shorter and the sensitivity increases.

[0092] Example 3

[0093] Investigating the influence of atomization temperature on the detection method

[0094] The nebulizer temperatures were set to 35°C, 40°C, 45°C, 50°C, and 60°C, respectively, and the other chromatographic conditions were the same as in Example 1.

[0095] Mixed related substance reference substance solution 2: Take 0.5 ml of the mixed related substance reference substance stock solution of Example 1, place it in a 2 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0096] The mixed related substance reference solution 2 was injected for testing, and the chromatogram was as follows Figure 3 to Figure 5 When the nebulizer temperature is within the range of 35℃~60℃, the peak shapes of related substances are good, the separation is good, the baseline of continuous injection is stable, and the signal response of related substances decreases with the increase of temperature, which can meet the sensitivity requirements of the control level of related substances in phospholipid-containing preparations.

[0097] Example 4

[0098] Investigate the influence of mobile phase A on the detection method

[0099] The mobile phase A was prepared as 10 mM ammonium formate buffered saline solution, pH 3.0, and the other chromatographic conditions were the same as condition 1 of Example 2. The mixed related substance reference substance stock solution of Example 1 was used for sample injection detection, and the peaks of each related substance were symmetrical and sharp, with a separation degree of more than 1.5.

[0100] Example 5

[0101] Recovery rate test

[0102] The flow rate was set to 1.1 ml / min, the column temperature was 37° C., the nebulizer temperature was 48° C., and the other chromatographic conditions were the same as those in Example 1. The system suitability solution injection test of Example 1 was used, and the peaks of the related substances were symmetrical and sharp, the separation degree reached more than 1.5, and the recoveries of LPA, palmitic acid and palmitoyl propanediol were 98.05%, 106.36% and 104.76%, respectively.

[0103] Example 6

[0104] The chromatographic column, mobile phase and gradient elution program of Example 2 were used, and the other chromatographic conditions were the same as those of Example 1. The mixed related substance reference solution 1 of Example 2 was injected for detection, and the chromatogram was as shown in FIG. Figure 6 This detection method can accurately detect related substances in phospholipid-containing preparations. LPC, LPA, palmitic acid and palmitoyl propanediol can all be effectively detected with good sensitivity and a relatively stable baseline after continuous sampling.

[0105] Comparative Example 1

[0106] Investigate the influence of mobile phase elution procedure on detection method

[0107] The mobile phase gradient elution program and isocratic elution program were set respectively, as shown in Tables 4 and 5 below, and the other chromatographic conditions were the same as in Example 4.

[0108] The mixed related substance reference substance stock solution of Example 1 was sampled and tested, and the chromatogram was as follows: Figure 7-8 As shown. Figure 7 It can be seen that after increasing the proportion of mobile phase B in the initial gradient, no LPA peak was observed. Figure 8 It can be seen that the retention time becomes shorter when isocratic elution is used, but the chromatographic peaks are split, which does not meet the detection requirements.

[0109] Table 4 Gradient elution program

[0110] Time / minute 0 5 15 20 25 A％ 20 10 10 20 20 B％ 80 90 90 80 80

[0111] Table 5 Isocratic elution program

[0112] Time / minute 0 25 A％ 10 10 B％ 90 90

[0113] The mobile phase gradient elution program is set as shown in Table 6, and the other chromatographic conditions are the same as those of the chromatographic column 1 in Example 1. The mixed related substance reference solution 1 of Example 2 was used for injection testing, and the LPA separation degree was low and could not be effectively separated from the adjacent chromatographic peaks.

[0114] Table 6 Gradient elution program

[0115] Time (minutes) A％ B％ C％ 0 40 60 0 20 10 90 0 30 10 90 0 40 0 0 100 50 0 0 100 55 40 60 0 65 40 60 0

[0116] Comparative Example 2

[0117] Investigating the influence of organic solvent in mobile phase on detection method

[0118] The mobile phase B was set to acetonitrile-methanol (80:20), and the other chromatographic conditions were the same as those in Example 2, Condition 3.

[0119] When the mixed related substance reference solution 1 of Example 2 was injected for detection, the peaks of LPC and LPA overlapped, and LPA could not be effectively separated from LPC.

[0120] Comparative Example 3

[0121] Investigating the impact of detectors on detection methods

[0122] Chromatographic column: Octadecylsilane bonded silica gel as filler, pore size

[0123] Mobile phase A: 5 mM ammonium acetate in water; Mobile phase B: 5 mM ammonium acetate in methanol;

[0124] Flow rate: 1.2 ml / min; column temperature: 25 °C; injection volume: 100 μl;

[0125] ELSD detector, drift tube temperature: 100 °C, carrier gas flow rate: 3.0 L / min;

[0126] The gradient elution program is shown in Table 7 below.

[0127] Table 7 Gradient elution program

[0128]

[0129]

[0130] The mixed related substance reference solution 2 of Example 3 was sampled and tested, and the chromatogram was as follows: Fig. 9 No signal response was detected in the ELSD detection mode, indicating that the response of these related substances on the ELSD detector was particularly poor and did not meet the product detection requirements.

[0131] The method of the present invention has been described through preferred embodiments, and it is obvious that those skilled in the art can modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to implement the technology of the present invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention.

Claims

1. A method for separating related substances from a phospholipid preparation, characterized in that: The separation method adopts high performance liquid chromatography, the chromatographic column adopts octadecylsilane bonded silica gel as filler, the mobile phase is mobile phase A, mobile phase B and optional mobile phase C, and the related substances include one or more of LPA, palmitic acid and palmitoyl propanediol.

2. The separation method according to claim 1, characterized in that The pore size of the chromatographic column is Best 3. The separation method according to claim 1 or 2, characterized in that The mobile phase A is a buffered saline solution, and the mobile phase B and mobile phase C are both organic solvents; preferably, the mobile phase B is acetonitrile, and the mobile phase C is methanol.

4. The separation method according to any one of claims 1 to 3, characterized in that The mobile phase A is a volatile buffered saline solution.

5. The separation method according to any one of claims 1 to 4, characterized in that The mobile phase A is selected from one or both of ammonium formate buffered saline solution and ammonium acetate buffered saline solution, preferably ammonium formate buffered saline solution.

6. The separation method according to any one of claims 1 to 5, characterized in that The buffer salt concentration in the mobile phase A is 10-50 mM, preferably 20-40 mM, more preferably 20-30 mM, more preferably 24-26 mM, and more preferably 25 mM.

7. The separation method according to any one of claims 1 to 6, characterized in that The pH of the mobile phase A is 2.5 to 3.5, preferably 2.8 to 3.2, and more preferably 3.

0.

8. The separation method according to any one of claims 1 to 7, characterized in that The detector used is an evaporative light scattering detector or an electrospray detector, preferably an electrospray detector; the atomizer temperature of the electrospray detector is 35-60°C, preferably 40-60°C, more preferably 45-55°C, more preferably 48-52°C, and more preferably 50°C.

9. The separation method according to any one of claims 1 to 8, which can be used for continuous detection and maintaining a stable baseline.

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