Method for analyzing and detecting related substances of distearoyl phosphatidyl sodium glycerol

Abstract

The invention discloses an analysis and detection method for related substances of sodium distearoyl phosphatidyl glycerol. A chromatographic column with octyl bonded silica gel as a filler is adopted; an ammonium acetate buffer solution for adjusting the pH value to 2.9-3.1 or an ammonium formate buffer solution for adjusting the pH value to 1.9-2.1 is used as a mobile phase A; and taking an ammonium acetate methanol solution, an ammonium formate methanol solution, a methanol solution of formic acid or methanol as a mobile phase B, and carrying out gradient elution. The high performance liquid chromatography electrospray detector method can be used for separating and detecting various impurities to be detected at the same time, the solvent is simple, and the used reagent is low in toxicity; the method disclosed by the invention is high in sensitivity and good in stability, and can be used for quantitatively and accurately detecting impurities in sodium stearoyl phosphatidyl glycerol.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis and relates to an analysis and detection method for related substances of sodium distearoylphosphatidylglycerol. Background Art

[0002] Sodium distearoylphosphatidylglycerol (DSPG-NA) is a phospholipid compound, the chemical name is 1,2-distearoyl-sn-glycero-3-phosphoglycerol sodium salt, CAS is 124011-52-5, the chemical structure is:

[0003] Sodium distearoylphosphatidylglycerol is mostly used in the preparation of liposomes, and is also used in the preparation of micelles, nanoparticles and other nanotechnologies.

[0004] Sodium distearoylphosphatidylglycerol has no ultraviolet absorption, and its related substances cannot be determined by high performance liquid chromatography-ultraviolet detection (HPLC-UV). At present, in the original import registration standard, the determination of related substances of this substance uses thin layer chromatography, which is divided into two system tests (the first method is to determine lysophosphatidylglycerol, phosphatidic acid, phosphatidylcholine and other unknown substances; the second method is to determine free fatty acids). Different developing agents are required for separation and determination. The test results are not intuitive enough, the test reagents are of many varieties, highly toxic, and the process operation is cumbersome. In addition, the thin layer chromatography method can only achieve limit inspection, and cannot perform quantitative detection. Summary of the invention

[0005] The purpose of the present invention is to provide a method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol.

[0006] The present invention has developed a high performance liquid chromatography combined with an electrospray ionization detector (HPLC-CAD) method for detecting related substances of sodium distearoylphosphatidylglycerol. The detection method established by the present invention can simultaneously determine the following multiple sodium distearoylphosphatidylglycerol impurities: Impurity I: Lysophosphatidylglycerol (LSPG-Na) The chemical name is 1.2-distearoyl-sn-glycero-3-phosphoglycerol sodium salt; CAS number is 326495-23-2; the chemical structure is as follows:

[0007] Impurity II: Stearic acid The chemical name is octadecanoic acid; CAS number is 57-11-4 The chemical structure is:

[0008] Impurity III: Phosphatidic acid (DSPA-Na) The chemical name is 1,2-distearoyl-sn-glycerol-3-phosphate sodium salt CAS is 108321-18-2 The chemical structure is:

[0009] Impurity IV: Phosphatidylcholine (DSPC) The chemical name is 1,2-distearoyl-sn-glycero-3-phosphocholine The English name is 1,2-Distearoyl-sn-glycero-3-phosphocholine CAS is 816-94-4 The molecular formula is C44H88NO8 P, The chemical structure is:

[0010] The chromatographic conditions of the present invention are: A chromatographic column with octyl bonded silica as filler was used; ammonium acetate buffer (adjusted to pH 2.9-3.1) or ammonium formate buffer (adjusted to pH 1.9-2.1) was used as mobile phase A; ammonium acetate methanol solution, ammonium formate methanol solution, formic acid methanol solution or methanol was used as mobile phase B; the gradient elution program was: 0min→10min, 23%~27% mobile phase A, 77%~73% mobile phase B; 10min→30min, 23%~27% mobile phase A, 77%~73% mobile phase B linearly changed to 12% mobile phase A, 88% mobile phase B; 30min→80min, 12% mobile phase A, 88% mobile phase B linearly changed to 8% mobile phase A, 92% mobile phase B; 80 min → 85 min, 8% mobile phase A, 92% mobile phase B; 85min→86min, 8% mobile phase A, 92% mobile phase B linearly changed to 23%~27% mobile phase A, 77%~73% mobile phase B; 86min→100min, 23%~27% mobile phase A, 77%~73% mobile phase B.

[0011] In order to optimize the above technical solutions, the specific limitations adopted also include: The nebulization temperature of the electrospray detector was 35~50 °C.

[0012] The analytical detection method can simultaneously separate and detect distearoylphosphatidylglycerol sodium and impurity I: lysophosphatidylglycerol, impurity II: stearic acid, impurity III: phosphatidic acid and impurity IV: phosphatidylcholine.

[0013] The chromatographic column adopts Luna ® 5μm C 8 (2) 100Å, 250×4.6mm.

[0014] The mobile phase A is 0.14-0.16 mol / L ammonium acetate buffer, the pH value is adjusted to 2.9-3.1 with acetic acid, or 0.09-0.11 mol / L ammonium formate buffer, the pH value is adjusted to 1.9-2.1 with formic acid; preferably, the mobile phase A is 0.15 mol / L ammonium acetate buffer, the pH value is adjusted to 3.0 with acetic acid.

[0015] The mobile phase B is 0.009-0.011 mol / L ammonium acetate methanol solution, or 0.009-0.011 mol / L ammonium formate methanol solution, or 0.09%-0.11% formic acid methanol solution; preferably, the mobile phase B is 0.010 mol / L ammonium acetate methanol solution.

[0016] The gradient elution program was: 0 min → 10 min, 25% mobile phase A, 75% mobile phase B; 10min→30min, 25% mobile phase A, 75% mobile phase B linearly changed to 12% mobile phase A, 88% mobile phase B; 30min→80min, 12% mobile phase A, 88% mobile phase B linearly changed to 8% mobile phase A, 92% mobile phase B; 80 min → 85 min, 8% mobile phase A, 92% mobile phase B; 85min→86min, 8% mobile phase A, 92% mobile phase B linearly changed to 25% mobile phase A, 75% mobile phase B; 86min→100min, 25% mobile phase A, 75% mobile phase B.

[0017] The test solution was prepared by weighing sodium distearoylphosphatidylglycerol, dissolving it in a solvent, and quantitatively diluting it to make a solution of 3.2~4.0 mg / ml; The column temperature is 28-32°C, and the detection flow rate is 0.9-1.1 ml / min. Preferably, the column temperature is 30°C, and the mobile phase flow rate is 1.0 ml / min.

[0018] The solvent is: tert-butyl alcohol-water, 45-55:45-55 v / v; the injection volume is 20-50 μl. Preferably, the injection volume is 50 μl.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention develops and optimizes a high performance liquid chromatography-electrospray ionization detector (HPLC-CAD) method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol, which can intuitively, effectively and accurately detect related substances of sodium distearoylphosphatidylglycerol (DSPG-NA).

[0020] The analytical detection method of the present invention can simultaneously separate and detect four impurities to be detected which are detected by the first method and the second method of two thin layer chromatography methods according to the registration standards of imported drugs.

[0021] The invention can effectively separate sodium distearoylphosphatidylglycerol and its impurities, and quantitatively detect related impurities in the sodium distearoylphosphatidylglycerol.

[0022] The detection reagent used in the present invention is simple, wherein ammonium acetate, acetic acid and methanol are used, all of which are low in toxicity. The method is suitable for popularization and application.

[0023] The method of the invention fills the gap of the current lack of an effective quantitative method suitable for the analysis and detection of related substances of sodium distearoylphosphatidylglycerol.

[0024] It has been verified that the method of the present invention has good specificity, high accuracy, high sensitivity and good stability, and can accurately detect impurities in sodium stearoylphosphatidylglycerol. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the HPLC spectrum of the spiked sample solution in Example 1.

[0026] Figure 2 The HPLC spectrum of the reference substance solution in Example 2.

[0027] Figure 3 The HPLC spectrum of the test solution in Example 2.

[0028] Figure 4 This is the HPLC spectrum of the control solution in Example 2.

[0029] Figure 5 This is the HPLC spectrum of the system suitability solution in Example 3.

[0030] Figure 6 This is the HPLC spectrum of the mixed solution in Example 4.

[0031] Figure 7 This is the HPLC spectrum of the mixed solution in Example 5.

[0032] Figure 8 This is the thin layer detection diagram of the first method in Comparative Example 1.

[0033] Fig. 9 This is the thin layer detection diagram of the second method in Comparative Example 1.

[0034] Fig.10 It is the HPLC chart of DSPG-Na in Comparative Example 2.

[0035] Fig.11 This is the HPLC chart of the mixed solution in Comparative Example 3.

[0036] Fig.12 This is the HPLC chart of the mixed solution in Comparative Example 4.

[0037] Fig.13 This is the HPLC chart of the mixed solution in Comparative Example 5. DETAILED DESCRIPTION

[0038] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0039] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0040] About the abbreviations in this application: DSPG-Na is the abbreviation of sodium distearoylphosphatidylglycerol; DSPC is the abbreviation for phosphatidylcholine; LSPG-Na is the abbreviation for lysophosphatidylglycerol; DSPA-Na is the abbreviation of phosphatidic acid.

[0041] The method of the present application uses a chromatographic column filled with octyl bonded silica gel, such as Luna ® 5μm C 8 (2) 100Å, 250×4.6mm or equivalent columns; Luna ® 5μm C 8 (2) 100Å is a product model, where (2) indicates the second generation C 8 Silane-based column, Å indicates the pore size of the column filling particles.

[0042] The solvent used in the following examples for preparing the test solution is a mixed solution of tert-butyl alcohol: water = 50:50 by volume.

[0043] Example 1

[0044] Instrument: Thermo Fisher U3000 equipped with electrospray ionization detector.

[0045] Chromatographic conditions: Luna ® 5μm C 8 (2) 100Å, 250×4.6mm; 0.15mol / L ammonium acetate buffer (pH adjusted to 3.0 with glacial acetic acid) as mobile phase A, 0.01mol / L ammonium acetate methanol solution as mobile phase B, flow rate of 1.0ml / min, CAD detector nebulization temperature of 35℃, column temperature of 30℃, injection plate temperature of 25℃, injection volume of 50μl; gradient elution according to the table below.

[0046] Table 1 Elution gradient method of Example 1

[0047] Mobile phase preparation: Mobile phase A: Add 11.56 g of ammonium acetate to 1 L of water and adjust the pH to 3.0 with glacial acetic acid.

[0048] Mobile phase B: Weigh 3.08 g of ammonium acetate into 4 L of methanol, dissolve and mix well.

[0049] Solution preparation: Lysophosphatidylglycerol (LSPG-Na) positioning solution: Weigh an appropriate amount of lysophosphatidylglycerol (LSPG-Na), add solvent to dissolve and dilute to make a solution of appropriate concentration.

[0050] Stearic acid positioning solution: Weigh an appropriate amount of stearic acid, add solvent to dissolve and dilute to make a solution of appropriate concentration.

[0051] Sodium distearoylphosphatidylglycerol (DSPG-Na) positioning solution: Weigh an appropriate amount of sodium distearoylphosphatidylglycerol (DSPG-Na), add solvent to dissolve and dilute to make a solution of appropriate concentration.

[0052] Phosphatidic acid (DSPA-Na) positioning solution: Weigh an appropriate amount of phosphatidic acid (DSPA-Na), add solvent to dissolve and dilute to make a solution of appropriate concentration.

[0053] Phosphatidylcholine (DSPC) localization solution: weigh an appropriate amount of phosphatidylcholine (DSPC), add solvent to dissolve and dilute to make a solution of appropriate concentration.

[0054] Spiked sample solution (system suitability solution): Take an appropriate amount of this product and add an appropriate amount of each impurity location solution, dissolve it with the solution and quantitatively dilute it to make a mixed solution containing about 36μg of lysophosphatidylglycerol (LSPG-Na), about 36μg of stearic acid, about 36μg of phosphatidic acid (DSPA-Na), about 36μg of phosphatidylcholine (DSPC), and about 3.6mg of distearoylphosphatidylglycerol sodium (DSPG-Na) per 1ml.

[0055] Method: Accurately measure 50 μl of the system suitability solution and each positioning solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0056] The results are shown in Table 2, and the spiked sample solutions are shown in Figure 1 .

[0057] Table 2 Separation test results

[0058] Example 2 Determination of Related Substances in Distearoylphosphatidylglycerol Sodium (DSPG-NA) Reference solution: Accurately weigh about 18 mg of lysophosphatidylglycerol (LSPG-Na) reference and about 18 mg of stearic acid reference, place in the same 25 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well, and make a mixed stock solution of LSPG-Na reference and stearic acid reference. Accurately weigh about 18 mg of phosphatidic acid (DSPA-Na) reference and about 18 mg of phosphatidylcholine (DSPC) reference, place in the same 50 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well, and make a mixed stock solution of DSPA-Na reference and DSPC reference. Accurately measure 0.5 ml of the mixed stock solution of LSPG-Na reference and stearic acid reference and 1.0 ml of the mixed stock solution of DSPA-Na reference and DSPC reference to the same 10 ml volumetric flask, dissolve and dilute to scale with solvent, shake well. Test solution: Accurately weigh about 36 mg of this product, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to scale. Control solution: Accurately measure 1.0 ml of the test solution, place it in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0059] Liquid chromatography analysis was performed according to the chromatographic conditions of Example 1. In the chromatogram of the test solution, if there were chromatographic peaks with the same retention time as the peaks of lysophosphatidylglycerol (LSPG-Na), stearic acid, phosphatidic acid (DSPA-Na), and phosphatidylcholine (DSPC), their contents were calculated by peak area using the external standard method, and the contents of other individual impurities were calculated by the self-control method.

[0060] For the determination of the reference solution of related substances, see Figure 2 , for the determination of the test solution of related substances, see Figure 3 , for the determination of the reference solution of related substances, see Figure 4 .

[0061] Embodiment 3: Referring to the experimental operation and condition parameters of Example 1, the flow rate, column temperature, pH value in mobile phase A and the ratio of the initial mobile phase and the batch of the chromatographic column in the chromatographic conditions were changed respectively, and other conditions and parameters remained unchanged, and the separation between the impurities and the main peak in the mixed spiked test solution was investigated. The results are shown in Table 3. The minimum separation is the minimum value of the separation between the main peak (DSPG-Na) and the known impurities and the adjacent impurities in the system suitability solution. The minimum value is 1.6, which is 27% of the initial B phase (organic phase). Figure 5 .

[0062] Table 3 Durability test results of related substances of distearoylphosphatidylglycerol sodium (DSPG-Na)

[0063] Note: The minimum resolution is the minimum resolution between the main peak (DSPG-Na) and known impurities and adjacent impurities in the system suitability solution.

[0064] Embodiment 4: Instrument: Thermo Fisher U3000 equipped with electrospray ionization detector.

[0065] Chromatographic conditions: Octylsilane bonded silica column (Luna ® 5μm C 8 (2) 100Å, 250×4.6mm); 0.10mol / L ammonium formate buffer (pH adjusted to 2.0 with formic acid) as mobile phase A, methanol as mobile phase B, flow rate of 1.0ml / min, CAD detector nebulization temperature of 35℃, column temperature of 30℃, injection volume of 20μl. Gradient elution was performed as shown in the table below.

[0066] Table 4 Elution gradient method of Example 4

[0067] Mobile phase preparation: Mobile phase A: Add 6.3 g of ammonium formate to 1 L of water and adjust the pH to 2.0 with formic acid.

[0068] Mobile phase B: methanol.

[0069] Solution preparation: Take LSPG-Na, stearic acid, DSPA-Na, DSPC and DSPG-Na, dissolve in solvent and quantitatively dilute to make a mixed solution containing about 0.12 mg of LSPG-Na, stearic acid, DSPA-Na, DSPC and about 0.5 mg of DSPG-Na per 1 ml, as the mixed solution, and inject. Figure 6 LSPG-Na, stearic acid, DSPA-Na, DSPG-Na and DSPC were eluted in sequence, and the separation degree met the requirements.

[0070] Embodiment 5: Instrument: Thermo Fisher U3000 equipped with electrospray ionization detector.

[0071] Chromatographic conditions: Octylsilane bonded silica column (Luna ® 5μm C 8 (2) 100Å, 250×4.6mm) or equivalent column; 0.10mol / L ammonium formate buffer (pH adjusted to 2.0 with formic acid) as mobile phase A, 0.1% formic acid in methanol as mobile phase B, flow rate of 1.0ml / min, CAD detector nebulization temperature of 35℃, column temperature of 30℃, injection volume of 20μl. Gradient elution according to the table below.

[0072] Table 5 Elution gradient method of Example 5

[0073] Mobile phase preparation: Mobile phase A: Add 6.3 g of ammonium formate to 1 L of water and adjust the pH to 2.0 with formic acid.

[0074] Mobile phase B: Take 1.0 ml of formic acid and add water to 1 L, mix well.

[0075] Solution preparation: Take LSPG-Na, stearic acid, DSPA-Na, DSPC and DSPG-Na, dissolve in solvent and quantitatively dilute to make a mixed solution containing about 0.12 mg of LSPG-Na, stearic acid, DSPA-Na, DSPC and about 0.5 mg of DSPG-Na per 1 ml, as the mixed solution, and inject. Figure 7 LSPG-Na, stearic acid, DSPA-Na, DSPG-Na and DSPC were eluted in sequence, and the separation degree met the requirements.

[0076] Comparative Example 1 Thin layer chromatography (registration standard for imported drugs, standard number JF20220011) The registration standard for imported drugs divides the above four known impurities into two thin layer chromatography methods (the first method is for the determination of lysophosphatidylglycerol, phosphatidic acid, phosphatidylcholine and other unknown substances; the second method is for the determination of free fatty acids) and uses different developing agents for separation and determination.

[0077] Method 1: Determination of lysophosphatidylglycerol, phosphatidic acid, phosphatidylcholine and other unknown substances The specific method is: The solvent was chloroform-methanol-water (65:25:4); The developing solvent was chloroform-methanol-water-concentrated ammonia solution (65:25:4:0.4); Copper sulfate phosphoric acid solution (color developer) is to take 8 ml of phosphoric acid, put it in a beaker with about 60 ml of water, shake it well, add 10 g of anhydrous copper sulfate, dissolve it, and add water to 100 ml; Solution preparation: Accurately weigh an appropriate amount of the product, dissolve and dilute with a solvent to prepare a solution containing 10 mg per 1 ml, as the test solution; accurately weigh an appropriate amount of the test solution, dilute with a solvent to prepare solutions containing approximately 0.025 mg, 0.05 mg and 0.1 mg per 1 ml, as control solution (1), control solution (2) and control solution (3); accurately weigh an appropriate amount of lysophosphatidylglycerol, phosphatidic acid and phosphatidylcholine reference substances, dissolve and dilute with a solvent to prepare a mixed solution containing 0.05 mg of each substance per 1 ml, as the reference substance mixed solution (1); take an equal volume of the reference substance mixed solution (1) and solvent and mix them to prepare the reference substance mixed solution (2).

[0078] Take 10 μl of each of the above solutions and spot them on the same silica gel G aluminum plate, place it in a chromatography cylinder with filter paper moistened with a developer on the inner wall, develop it, and let it dry. Spray it with copper sulfate phosphoric acid solution and heat it at 170°C for 10 minutes. Observe the spot colors of the reference mixed solution, the test solution and the reference solution (1), the reference solution (2) and the reference solution (3). If the test solution shows impurity spots, compare them with the corresponding impurity spots and calculate their content. Take 3 batches of the test sample for determination. The results are shown in the following table and Figure 8 .

[0079] Table 6 Detection results of lysophosphatidylglycerol, phosphatidic acid, phosphatidylcholine and other unknown substances in DSPG-Na

[0080] Method 2: Determination of free fatty acids The specific method is: The solvent was chloroform-methanol-water (65:25:4); The developing solvent was n-hexane-ether-glacial acetic acid (70:30:1); Copper sulfate phosphoric acid solution (color developer) is to take 8 ml of phosphoric acid, put it in a beaker with about 60 ml of water, shake it well, add 10 g of anhydrous copper sulfate, dissolve it, and add water to 100 ml; Solution preparation: Accurately weigh an appropriate amount of stearic acid reference substance, dissolve it with a solvent and quantitatively dilute it to make solutions containing 0.03mg and 0.01mg per 1ml, respectively, as reference substance solutions; accurately weigh an appropriate amount of this product, dissolve it with a solvent and quantitatively dilute it to make a solution containing 10mg per 1ml, as test solution; accurately weigh an appropriate amount of this product, dissolve it with a reference substance solution (0.03mg / ml) and quantitatively dilute it to make a solution containing 10mg per 1ml, as spiked test solution; aspirate 20ul of each sample of the above reference substance solution, test solution and spiked test solution at 0 o'clock and at room temperature for 18 hours, respectively, and apply them in strips on the same silica gel Gs aluminum plate, develop them in a chromatography cylinder with filter paper moistened with a developer on the inner wall, and let them dry. Spray with copper sulfate phosphoric acid solution and heat at 170°C for 10 minutes. Observe the spot color of the reference substance solution, test solution and spiked test solution. The results are shown in the table below and the typical spectrum is shown in Fig. 9 .

[0081] Table 7 Free fatty acid test results of DSPG-Na

[0082] Comparative Example 2 Literature Method Reference: "HPLC-charged aerosol detector (CAD) detection method for determination of phospholipid content in liposomes" from "Chinese Journal of Pharmaceutical Sciences" Vol. 42 No. 23, December 2007 Instrument: Thermo Fisher U3000 equipped with electrospray ionization detector Chromatographic conditions: The chromatographic column is Thermo scientific Hypersil GOLD C18 4.6*250mm, 5um; water is used as mobile phase A, methanol is used as mobile phase B; CAD atomization temperature is 35℃; column temperature is 30℃; flow rate is 1ml / min; injection volume is 10ul, and gradient elution is performed as shown in the following table: Table 8 Elution gradient table in comparative example 2

[0083] Solution preparation: The solvent is water-tert-butyl alcohol (1:1), weigh about 24 mg of DSPG-Na and put it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale and shake well, take 10 ul and inject it into the chromatograph, and record the chromatogram. The results are shown in Fig.10 , DSPG-Na peak shape tailing.

[0084] The following comparative examples 3-5 are high performance liquid chromatography combined with electrospray ionization detector (HPLC-CAD) methods using other conditions; these comparative examples are not exhaustive, and the present application has finally concluded through the exploration of various experimental conditions and parameters that the methods established in Examples 1, 4, and 5 are the most suitable methods for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol.

[0085] Comparative Example 3 Instrument: Thermo Fisher U3000 equipped with electrospray detector.

[0086] Chromatographic conditions: Octylsilane bonded silica column (Luna® 5μm C8(2) 100Å, 250×4.6mm); 0.1mol / L ammonium acetate solution (pH adjusted to 3.0 with acetic acid): methanol (75:25) as mobile phase A, methanol as mobile phase B, flow rate of 1.0ml / min, CAD detector nebulization temperature of 35℃, column temperature of 30℃, injection volume of 50μl. Injection plate temperature of 25℃, gradient elution according to the table below.

[0087] Table 9 Elution gradient method of comparative example 3

[0088] Preparation of mixed solution [adding the formulation ingredients, i.e., excipients and dipalmitoylphosphatidylcholine (DPPC) and related impurities, lysophosphatidylcholine (LPPC) and palmitic acid, to DSPG-Na and related impurities]: Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoylphosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC solvent [water-tert-butyl alcohol (1:1)] were dissolved and quantitatively diluted to prepare a mixed solution containing 14 mg of mannitol, about 25 μg of LPPC, about 36 μg of LSPG-Na, about 16 μg of palmitic acid, about 36 μg of stearic acid, about 36 μg of phosphatidic acid, about 36 μg of phosphatidylcholine, about 0.8 mg of DPPC, and about 3.6 mg of DSPG-Na per 1 ml. Injection. Results are shown in Fig.11 Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoylphosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC eluted in sequence, and the DSPA-Na peak and the DSPC peak overlapped and were not separated.

[0089] Comparative Example 4 Instrument: Thermo Fisher U3000 equipped with electrospray detector.

[0090] Chromatographic conditions: Octylsilane bonded silica column (Luna® 5μm C8(2) 100Å, 250×4.6mm); 0.1mol / L ammonium acetate solution (pH adjusted to 3.0 with acetic acid): methanol (75:25) as mobile phase A, 0.1mol / L ammonium acetate solution (pH adjusted to 3.0 with acetic acid): methanol (5:95) as mobile phase B, flow rate of 1.0ml / min, CAD detector nebulization temperature of 35℃, column temperature of 30℃, injection volume of 50μl. Injection plate temperature of 25℃, gradient elution according to the table below.

[0091] Table 10 Elution gradient method of comparative example 4

[0092] Mixed solution preparation: Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoylphosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC solvent [water-tert-butyl alcohol (1:1)] were dissolved and quantitatively diluted to prepare a mixed solution containing 14 mg of mannitol, about 25 μg of LPPC, about 36 μg of LSPG-Na, about 16 μg of palmitic acid, about 36 μg of stearic acid, about 36 μg of phosphatidic acid, about 36 μg of phosphatidylcholine, about 0.8 mg of DPPC, and about 3.6 mg of DSPG-Na per 1 ml. Injection. Results are shown in Fig.12 Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoylphosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC eluted in sequence, and the peak separation between the DSPA-Na peak and the DSPC peak did not meet the requirements. After the mixed solution DSPG-Na, there were only two impurity peaks, DSPA-Na and DSPC, and the unknown impurities of DSPG-Na were not eluted.

[0093] Comparative Example 5 Instrument: Thermo Fisher U3000 equipped with electrospray detector.

[0094] Chromatographic conditions: Octylsilane bonded silica column (Luna® 5μm C8(2) 100Å, 250×4.6mm); 0.1mol / L ammonium acetate solution (pH adjusted to 3.0 with acetic acid): methanol (75:25) as mobile phase A, 0.1mol / L ammonium acetate solution (pH adjusted to 3.0 with acetic acid): methanol (5:95) as mobile phase B, flow rate of 1.0ml / min, CAD detector nebulization temperature of 35℃, column temperature of 30℃, injection volume of 50μl. Injection plate temperature of 25℃, gradient elution according to the table below.

[0095] Table 11 Elution gradient method of comparative example 5

[0096] Mixed solution preparation: Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoylphosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC solvent [water-tert-butyl alcohol (1:1)] were dissolved and quantitatively diluted to prepare a mixed solution containing 14 mg of mannitol, about 25 μg of LPPC, about 36 μg of LSPG-Na, about 16 μg of palmitic acid, about 36 μg of stearic acid, about 36 μg of phosphatidic acid, about 36 μg of phosphatidylcholine, about 0.8 mg of DPPC, and about 3.6 mg of DSPG-Na per 1 ml. Injection. Results are shown in Fig.13 Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoylphosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC eluted in sequence. The peak separation between the DSPA-Na peak and the DSPC peak did not meet the requirements. However, there were 3 impurity peaks after DSPG-Na, which could elute the unknown impurities of DSPG-Na.

[0097] There are few high-performance liquid chromatography methods for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol. Currently, there are only a very small number of public schemes, such as the literature method of "HPLC-charged aerosol detector (CAD) detection method for determining the phospholipid content in liposomes" in Comparative Example 2, but the peak shape is tailing and the measurement effect is not good, which shows that the conditions for high-performance liquid chromatography for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol are difficult to obtain. This application also confirms this through the exploration of various experimental conditions and parameters.

[0098] The present invention has developed a detection method for sodium distearoylphosphatidylglycerol and its related substances through a large amount of research work, and solved various problems in the detection of related substances of the substance. The analytical detection method of the present invention can simultaneously separate and detect sodium distearoylphosphatidylglycerol and impurity I: lysophosphatidylglycerol, impurity II: stearic acid, impurity III: phosphatidic acid and impurity IV: phosphatidylcholine.

[0099] It can be seen from the test results of the embodiments and comparative examples that the detection and analysis method proposed by the present invention is more intuitive, and four known impurities and other unknown impurities in a sample can be detected using one chromatographic condition.

[0100] And after method verification, the specificity, detection limit and quantification limit, linearity, accuracy and durability of the method of the present invention have all passed the requirements of method verification, and can better and more conveniently realize the detection and control of DSPG-Na related substances.

[0101] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol, characterized in that: High performance liquid chromatography combined with an electrospray ionization detector was used for detection, and the chromatographic conditions were as follows: A chromatographic column with octyl bonded silica as filler was used; ammonium acetate buffer adjusted to pH 2.9-3.1 or ammonium formate buffer adjusted to pH 1.9-2.1 was used as mobile phase A; ammonium acetate methanol solution, ammonium formate methanol solution, formic acid methanol solution or methanol was used as mobile phase B; the gradient elution program was: 0min→10min, 23%~27% mobile phase A, 77%~73% mobile phase B; 10min→30min, 23%~27% mobile phase A, 77%~73% mobile phase B linearly changed to 12% mobile phase A, 88% mobile phase B; 30min→80min, 12% mobile phase A, 88% mobile phase B linearly changed to 8% mobile phase A, 92% mobile phase B; 80 min → 85 min, 8% mobile phase A, 92% mobile phase B; 85min→86min, 8% mobile phase A, 92% mobile phase B linearly changed to 23%~27% mobile phase A, 77%~73% mobile phase B; 86min→100min, 23%~27% mobile phase A, 77%~73% mobile phase B.

2. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The nebulization temperature of the electrospray detector was 35~50 °C.

3. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The analytical detection method can simultaneously separate and detect distearoylphosphatidylglycerol sodium and impurity I: lysophosphatidylglycerol, impurity II: stearic acid, impurity III: phosphatidic acid and impurity IV: phosphatidylcholine.

4. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The chromatographic column adopts Luna ® 5μm C8(2)100Å, 250×4.6mm.

5. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The mobile phase A is 0.14-0.16 mol / L ammonium acetate buffer, the pH value of which is adjusted to 2.9-3.1 with acetic acid, or 0.09-0.11 mol / L ammonium formate buffer, the pH value of which is adjusted to 1.9-2.1 with formic acid.

6. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The mobile phase B is 0.009-0.011 mol / L ammonium acetate methanol solution, or 0.009-0.011 mol / L ammonium formate methanol solution, or 0.09%-0.11% formic acid methanol solution.

7. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The gradient elution program was: 0 min → 10 min, 25% mobile phase A, 75% mobile phase B; 10min→30min, 25% mobile phase A, 75% mobile phase B linearly changed to 12% mobile phase A, 88% mobile phase B; 30min→80min, 12% mobile phase A, 88% mobile phase B linearly changed to 8% mobile phase A, 92% mobile phase B; 80 min → 85 min, 8% mobile phase A, 92% mobile phase B; 85min→86min, 8% mobile phase A, 92% mobile phase B linearly changed to 25% mobile phase A, 75% mobile phase B; 86min→100min, 25% mobile phase A, 75% mobile phase B.

8. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The test solution is prepared by weighing sodium distearoylphosphatidylglycerol, dissolving it in a solvent and quantitatively diluting it to make a solution of 3.2~4.0 mg / ml.

9. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The column temperature is 28~32°C; the detection flow rate is 0.9~1.1ml / min.

10. The method for analyzing and detecting related substances of sodium distearoylphosphatidylglycerol according to claim 1, characterized in that: The solvent is: tert-butyl alcohol-water, 45~55:45~55 v / v; the injection volume is 20~50μl.

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