CN119125393A - Analysis and detection methods of related substances of sodium distearoylphosphatidylglycerol - Google Patents
Analysis and detection methods of related substances of sodium distearoylphosphatidylglycerol Download PDFInfo
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Abstract
The invention discloses an analysis and detection method of distearyl phosphatidyl sodium glycerate related substances, which adopts a chromatographic column with octyl bonded silica gel as a filler, uses an ammonium acetate buffer solution to adjust the pH value to 2.9-3.1 or an ammonium formate buffer solution to adjust the pH value to 1.9-2.1 as a mobile phase A, and uses an ammonium acetate methanol solution, an ammonium formate methanol solution, a methanol solution of formic acid or methanol as a mobile phase B to perform gradient elution. The high performance liquid chromatography electrospray detector method can simultaneously separate and detect various impurities to be detected, has simple solvent and low toxicity of the used reagent, has high sensitivity and good stability, and can quantitatively and accurately detect the impurities in the sodium stearoyl phosphatidyl glycerol.
Description
Technical Field
The invention belongs to the technical field of chemical analysis, and relates to an analysis and detection method for distearoyl phosphatidyl sodium glycerate related substances.
Background
Distearoyl phosphatidyl sodium glycerate (DSPG-NA) is a phospholipid compound, the chemical name is 1, 2-distearoyl-sn-glycero-3-phosphoglycerate sodium salt, CAS is 124011-52-5, and the chemical structural formula is:
The distearoyl phosphatidyl sodium glycerolate is mostly used for preparing liposome, and is also applied to nano-technologies such as micelle, nano-particle and the like.
Sodium distearoyl phosphatidyl glycerol does not have ultraviolet absorption and related substances cannot be measured by high performance liquid chromatography-ultraviolet detection (HPLC-UV). At present, in the original import registration standard, the related substance measurement of the substance is divided into two systems for detection (the first method is to measure lysophosphatidylglycerol, phosphatidic acid, phosphatidylcholine and other unknown substances, the second method is to measure free fatty acid), and the separation and measurement are needed by using different developing agents, so that the detection result is not intuitive, the number of detection reagents is large, the toxicity is large, the process operation is complicated, and only the limit detection can be realized by the thin layer chromatography measurement method, and the quantitative detection cannot be performed.
Disclosure of Invention
The invention aims to provide an analysis and detection method for distearyl phosphatidyl sodium glycerolate related substances.
The invention develops a high performance liquid chromatography combined electrospray detector method (HPLC-CAD) for detecting related substances of distearoyl phosphatidyl sodium, and the detection method established by the invention can simultaneously detect the following multiple distearoyl phosphatidyl sodium impurities:
impurity I lysophosphatidylglycerol (LSPG-Na)
The chemical name is 1.2-distearoyl-sn-glycero-3-phosphoglycerate sodium salt, CAS is 326495-23-2, and the chemical structural formula is as follows:
Impurity II stearic acid
The chemical name is octadecanoic acid, and CAS is 57-11-4
The chemical structural formula is as follows:
Impurity III phosphatidic acid (DSPA-Na)
The chemical name is 1, 2-distearoyl-sn-glycero-3-phosphate sodium salt
CAS 108321-18-2
The chemical structural formula is as follows:
Impurity IV phosphatidylcholine (DSPC)
Chemical name is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine
English name 1, 2-Distearoyl-sn-glycero-3-phosphocholine
CAS 816-94-4
The molecular formula is C44H88NO 8P,
The chemical structural formula is as follows:
The chromatographic conditions of the invention are:
The method adopts a chromatographic column with octyl bonded silica gel as a filler, adopts an ammonium acetate buffer solution (with the pH value adjusted to be 2.9-3.1) or an ammonium formate buffer solution (with the pH value adjusted to be 1.9-2.1) as a mobile phase A, adopts an ammonium acetate methanol solution, an ammonium formate methanol solution, a methanol solution of formic acid or methanol as a mobile phase B, and comprises the following steps of:
0 min-10 min,23% -27% of mobile phase A and 77% -73% of mobile phase B;
10 min-30 min,23% -27% of mobile phase A and 77% -73% of mobile phase B are linearly changed into 12% of mobile phase A and 88% of mobile phase B;
30 min-80 min,12% mobile phase A and 88% mobile phase B are linearly changed into 8% mobile phase A and 92% mobile phase B;
80 min-85 min,8% mobile phase A, 92% mobile phase B;
85 min-86 min, wherein 8% of mobile phase A and 92% of mobile phase B are linearly changed into 23% -27% of mobile phase A and 77% -73% of mobile phase B;
86 min-100 min,23% -27% of mobile phase A and 77% -73% of mobile phase B.
In order to optimize the technical scheme, the specific limitations adopted further comprise:
the atomization temperature of the electrospray detector is 35-50 ℃.
The analysis and detection method can simultaneously separate and detect distearoyl phosphatidyl glycerol sodium and impurities I, II, III, IV and IV, wherein the impurities I and II are lysophosphatidyl glycerol, stearic acid and phosphatidic acid.
The chromatographic column uses Luna ®5μm C8 (2) 100 a, 250 x 4.6mm.
The mobile phase A is an ammonium acetate buffer solution with the concentration of 0.14-0.16 mol/L, the pH value is regulated to 2.9-3.1 by acetic acid, or an ammonium formate buffer solution with the concentration of 0.09-0.11 mol/L, the pH value is regulated to 1.9-2.1 by formic acid, and preferably, the mobile phase A is an ammonium acetate buffer solution with the concentration of 0.15 mol/L, and the pH value is regulated to 3.0 by acetic acid.
The mobile phase B adopts 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 0.010 mol/L ammonium acetate methanol solution.
The gradient elution procedure was:
0 min-10 min,25% mobile phase A, 75% mobile phase B;
10 min-30 min,25% of mobile phase A and 75% of mobile phase B are linearly changed into 12% of mobile phase A and 88% of mobile phase B;
30 min-80 min,12% mobile phase A and 88% mobile phase B are linearly changed into 8% mobile phase A and 92% mobile phase B;
80 min-85 min,8% mobile phase A, 92% mobile phase B;
85 min-86 min,8% mobile phase A and 92% mobile phase B are linearly changed into 25% mobile phase A and 75% mobile phase B;
86 min-100 min,25% mobile phase A, 75% mobile phase B.
The preparation of the sample solution is to weigh distearoyl phosphatidyl sodium glycerate, dissolve with solvent and dilute quantitatively to prepare 3.2-4.0 mg/ml solution;
the column temperature is 28-32 ℃, and the detection flow rate is 0.9-1.1 ml/min. Preferably, the column temperature is 30℃and the mobile phase flow rate is 1.0ml/min.
The solvent is tert-butanol-water, 45-55:45-55 v/v; the sample injection amount is 20-50 μl. Preferably, the sample is introduced in an amount of 50. Mu.l.
Compared with the prior art, the invention has the beneficial effects that:
the invention develops and optimizes the high performance liquid chromatography-electrospray detector method (HPLC-CAD) for analyzing and detecting distearyl phosphatidyl sodium glycerate related substances, and can intuitively, effectively and accurately detect distearyl phosphatidyl sodium glycerate (DSPG-NA) related substances.
The analysis and detection method can separate and detect 4 impurities to be detected simultaneously, wherein the 4 impurities to be detected are detected by a first method and a second method of two thin layer chromatography according to the imported drug registration standard.
The invention can effectively separate distearoyl phosphatidyl sodium glycerate and impurities thereof, and quantitatively detect related impurities in distearoyl phosphatidyl sodium glycerate.
The detection reagent adopted by the invention is simple, wherein ammonium acetate, acetic acid, methanol and the like are adopted, and the method is low in toxicity and suitable for popularization and application.
The method of the invention fills the blank of the prior effective quantitative method which is suitable for the analysis and detection of distearyl phosphatidyl sodium related substances.
Proved by verification, the method provided by the invention has the advantages of good specificity, high accuracy, high sensitivity and good stability, and can accurately detect the impurities in the sodium stearyl phosphatidyl glycerol.
Drawings
FIG. 1 is an HPLC chart of a standard sample solution for use in example 1.
FIG. 2 is an HPLC chart of the control solution in example 2.
FIG. 3 is an HPLC chart of a sample solution in example 2.
FIG. 4 is an HPLC chart of the control solution in example 2.
FIG. 5 is a HPLC chart of a system applicability solution in example 3.
FIG. 6 is an HPLC chart of the mixed solution in example 4.
FIG. 7 is an HPLC chart of the mixed solution in example 5.
FIG. 8 is a thin layer test chart of the first method in comparative example 1.
FIG. 9 is a thin layer test chart of the second method in comparative example 1.
FIG. 10 is an HPLC chart of DSPG-Na of comparative example 2.
FIG. 11 is an HPLC chart of the mixed solution in comparative example 3.
FIG. 12 is an HPLC chart of the mixed solution in comparative example 4.
Fig. 13 is an HPLC diagram of the mixed solution in comparative example 5.
Detailed Description
The above-described matters of the present invention will be further described in detail by way of examples, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples, and all techniques realized based on the above-described matters of the present invention are within the scope of the present invention.
The experimental methods used in the examples below are conventional methods, and the reagents, methods and apparatus used are conventional in the art, unless otherwise indicated.
Abbreviations for abbreviations in the present application:
DSPG-Na is an abbreviation for sodium distearoyl phosphatidyl glycerol;
DSPC is an abbreviation for phosphatidylcholine;
LSPG-Na is an abbreviation for lysophosphatidylglycerol;
DSPA-Na is an abbreviation for phosphatidic acid.
The method adopts a chromatographic column with octyl bonded silica gel as a filler, such as a chromatographic column of Luna ®5μm C8 (2) 100A, 250 multiplied by 4.6mm or equivalent performance, wherein Luna ®5μm C8 (2) 100A is a commodity model, wherein (2) represents a2 nd generation C 8 silane-based column, and A represents the pore size of filler particles of the chromatographic column.
The test solutions used in the following examples were prepared as mixed solutions of t-butanol and water=50:50 by volume.
Example 1
Instrument sameifeu 3000 is equipped with an electrospray detector.
The chromatographic conditions were Luna ®5μm C8 (2) 100A, 250X 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 atomization temperature of 35℃and column temperature of 30℃and sample tray temperature of 25℃and sample volume of 50. Mu.l, and gradient elution according to the following table.
TABLE 1 elution gradient mode for example 1
Mobile phase preparation:
Mobile phase a ammonium acetate 11.56g was taken and added to 1L water and pH adjusted to 3.0 with glacial acetic acid.
And (3) weighing 3.08g to 4L of ammonium acetate in methanol, and dissolving and uniformly mixing to obtain the mobile phase B.
Preparing a solution:
Dissolving and diluting lysophosphatidyl glycerol (LSPG-Na) with solvent to obtain solution with proper concentration.
And (3) stearic acid positioning solution, namely weighing a proper amount of stearic acid, and dissolving and diluting the stearic acid by adding a solvent to prepare a solution with proper concentration.
And (3) weighing a proper amount of distearoyl phosphatidyl sodium glycerate (DSPG-Na), and dissolving and diluting the distearoyl phosphatidyl sodium glycerate (DSPG-Na) by using a solvent to prepare a solution with proper concentration.
The phosphatidic acid (DSPA-Na) locating solution is prepared through weighing right amount of phosphatidic acid (DSPA-Na), dissolving in solvent and diluting.
And (3) phosphatidylcholine (DSPC) positioning solution, namely weighing a proper amount of phosphatidylcholine (DSPC), and adding a solvent for dissolving and diluting to prepare a solution with proper concentration.
Adding standard sample solution (system applicability solution), adding appropriate amount of the sample, adding appropriate amount of each impurity positioning solution, dissolving with the solution, and quantitatively diluting to obtain mixed solution containing lysophosphatidylglycerol (LSPG-Na) about 36 μg, stearic acid about 36 μg, phosphatidic acid (DSPA-Na) about 36 μg, phosphatidylcholine (DSPC) about 36 μg, distearoyl phosphatidylsodium glycerate (DSPG-Na) about 3.6mg per 1 ml.
The method comprises precisely measuring 50 μl of each of the system applicability solution and each of the positioning solutions, respectively injecting into a liquid chromatograph, and recording the chromatogram.
The results are shown in Table 2, and the solution of the added standard sample is shown in FIG. 1.
TABLE 2 separation test results
Example 2
Determination of distearoyl sodium phosphatidyl glycerol (DSPG-NA) related substances
The control solution is prepared by precisely weighing about 18mg of lysophosphatidyl glycerol (LSPG-Na) control and about 18mg of stearic acid control, placing into a same 25ml volumetric flask, adding solvent, dissolving, diluting to scale, shaking, and mixing to obtain mixed stock solution of LSPG-Na control and stearic acid control. Precisely weighing about 18mg of phosphatidic acid (DSPA-Na) reference substance and about 18mg of phosphatidylcholine (DSPC) reference substance, placing into a same 50ml volumetric flask, adding solvent to dissolve and dilute to scale, and shaking to obtain DSPA-Na reference substance and DSPC reference substance mixed stock solution. Precisely measuring 0.5ml of mixed stock solution of LSPG-Na reference substance and stearic acid reference substance and 1.0ml of mixed stock solution of DSPA-Na reference substance and DSPC reference substance into the same 10ml measuring flask, dissolving and diluting to scale with solvent, and shaking. The sample solution is precisely weighed to be about 36mg, put into a 10ml volumetric flask, dissolved by adding solvent and diluted to the scale. Shaking up. The control solution is prepared by precisely measuring 1.0ml of the sample solution, placing the sample solution into a 50ml measuring flask, diluting the sample solution to a scale with a solvent, and shaking the sample solution uniformly.
Liquid chromatography was performed under the chromatographic conditions of example 1, and the content of each of the sample solutions was calculated as the peak area by the external standard method and the content of each of the other individual impurities was calculated as the self-contrast method, for example, as the chromatographic peaks corresponding to the retention times of lysophosphatidylglycerol (LSPG-Na), stearic acid, phosphatidic acid (DSPA-Na) and phosphatidylcholine (DSPC).
The measurement of the reference substance solution is shown in FIG. 2, the measurement of the reference substance solution is shown in FIG. 3, and the measurement of the reference substance solution is shown in FIG. 4.
Example 3:
with reference to the experimental operation and condition parameters of example 1, the flow rate in chromatographic conditions, column temperature, pH value in mobile phase A, and the ratio of the initial mobile phase and batch of chromatographic columns were respectively changed, and other conditions and parameters were unchanged, and the separation between impurities and main peaks between solutions in the mixed and labeled sample was examined. The results are shown in Table 3, where the minimum degree of separation is the minimum of the main peak (DSPG-Na) in the system applicability solution and the degree of separation between the known impurity and the adjacent impurity, and the minimum is 1.6, which is 27% of the initial B phase (organic phase), as shown in FIG. 5.
TABLE 3 durability test results of distearoyl phosphatidyl sodium glycerate (DSPG-Na) related substances
Note that the minimum degree of separation is the minimum of the separation between a known impurity and an adjacent impurity in the main peak (DSPG-Na) in the system-applicable solution.
Example 4:
instrument sameifeu 3000 is equipped with an electrospray detector.
The chromatographic conditions were a octyl silane bonded silica gel column (Luna ®5μm C8 (2) 100 a, 250X 4.6 mm), 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 atomizing temperature of 35℃and column temperature of 30℃and sample injection volume of 20. Mu.l. The elution was performed gradient in the following table.
TABLE 4 elution gradient mode for example 4
Mobile phase preparation:
Mobile phase a ammonium formate 6.3g was taken and added to 1L water and pH adjusted to 2.0 with formic acid.
Mobile phase B, methanol.
Preparing a solution:
Dissolving LSPG-Na, stearic acid, DSPA-Na, DSPC and DSPG-Na in solvent, quantitatively diluting to obtain mixed solution containing about 0.12mg of LSPG-Na, stearic acid, DSPA-Na and DSPC and about 0.5mg of DSPG-Na per 1ml, and sampling. The results are shown in FIG. 6.LSPG-Na, stearic acid, DSPA-Na, DSPG-Na and DSPC sequentially show peaks, and the separation degree meets the requirements.
Example 5:
instrument sameifeu 3000 is equipped with an electrospray detector.
The chromatographic conditions are a octyl silane bonded silica gel column (Luna ®5μm C8 (2) 100 a, 250X 4.6 mm) or a chromatographic column with equivalent performance, 0.10mol/L ammonium formate buffer (formic acid is used for adjusting the pH value to 2.0) is used as a mobile phase A, 0.1% formic acid methanol solution is used as a mobile phase B, the flow rate is 1.0ml/min, the atomization temperature of a CAD detector is 35 ℃, the column temperature is 30 ℃, and the sample injection volume is 20 mu L. The elution was performed gradient in the following table.
TABLE 5 elution gradient mode for example 5
Mobile phase preparation:
Mobile phase a ammonium formate 6.3g was taken and added to 1L water and pH adjusted to 2.0 with formic acid.
And (3) taking 1.0ml of formic acid, adding water to 1L of the formic acid, and uniformly mixing the formic acid and the water.
Preparing a solution:
Dissolving LSPG-Na, stearic acid, DSPA-Na, DSPC and DSPG-Na in solvent, quantitatively diluting to obtain mixed solution containing about 0.12mg of LSPG-Na, stearic acid, DSPA-Na and DSPC and about 0.5mg of DSPG-Na per 1ml, and sampling. The results are shown in FIG. 7.LSPG-Na, stearic acid, DSPA-Na, DSPG-Na and DSPC sequentially show peaks, and the separation degree meets the requirement.
Comparative example 1 thin layer chromatography (imported drug registration Standard, standard No. JF 20220011)
Inlet drug registration standards the 4 known impurities were separated into two thin layer chromatographs (first method for measuring lysophosphatidylglycerol, phosphatidic acid, phosphatidylcholine and other unknowns; second method for measuring free fatty acids) using different developing agents for separation and measurement.
First method of measuring lysophosphatidylglycerol, phosphatidic acid, phosphatidylcholine and other unknowns
The specific method comprises the following steps:
The solvent is chloroform-methanol-water (65:25:4);
The developing agent is chloroform-methanol-water-concentrated ammonia solution (65:25:4:0.4);
the copper sulfate phosphoric acid solution (color developing agent) is prepared by taking 8ml of phosphoric acid, placing in a beaker with about 60ml of water added, shaking uniformly, adding 10g of anhydrous copper sulfate, dissolving, and adding water to 100ml;
preparing a solution:
Precisely weighing a proper amount of the product, dissolving and diluting the product by using a solvent to prepare a solution containing 10mg of each 1ml of the product as a test solution, precisely weighing a proper amount of the test solution, respectively diluting the solution by using the solvent to prepare solutions containing 0.025mg, 0.05mg and 0.1mg of each 1ml of the product as a control solution (1), a control solution (2) and a control solution (3), precisely weighing a proper amount of lysophosphatidylglycerol, phosphatidic acid and phosphatidylcholine reference substances, dissolving and diluting the reference solution by using the solvent to prepare mixed solutions containing 0.05mg of each 1ml of the product as a reference mixed solution (1), and mixing the reference mixed solution (1) and the solvent in equal volumes to prepare a reference mixed solution (2).
Respectively sucking 10 μl of the above solutions, respectively spotting on the same silica gel G aluminum plate, placing into a chromatographic cylinder with inner wall stuck with developer wetting filter paper, spreading, and air drying. Sprayed with a copper sulphate phosphoric acid solution and heated at 170 ℃ for 10 minutes. And (3) observing the mixed solution of the reference substances, the test substance solution and the reference solution (1), and comparing the spot colors of the reference solution (2) with those of the reference solution (3), wherein the contents of the test substance solution such as impurity spots and corresponding impurity spots are calculated. Test pieces 3 were taken and tested in the following table and fig. 8.
TABLE 6 detection results of lysophosphatidylglycerol, phosphatidic acid, phosphatidylcholine and other unknowns of DSPG-Na
Second method of measuring free fatty acid
The specific method comprises the following steps:
The solvent is chloroform-methanol-water (65:25:4);
The developing agent is n-hexane-diethyl ether-glacial acetic acid (70:30:1);
the copper sulfate phosphoric acid solution (color developing agent) is prepared by taking 8ml of phosphoric acid, placing in a beaker with about 60ml of water added, shaking uniformly, adding 10g of anhydrous copper sulfate, dissolving, and adding water to 100ml;
preparing a solution:
Precisely weighing a proper amount of stearic acid reference substance, dissolving and quantitatively diluting with a solvent to prepare solutions containing 0.03mg and 0.01mg in each 1ml of the reference substance solution respectively, precisely weighing a proper amount of the reference substance, dissolving and quantitatively diluting with a solvent to prepare a solution containing 10mg in each 1ml of the reference substance solution as a test substance solution, precisely weighing a proper amount of the reference substance, dissolving and quantitatively diluting with a reference substance solution (0.03 mg/ml) to prepare a solution containing 10mg in each 1ml of the reference substance solution as a standard test substance solution, respectively sucking the reference substance solution, the test substance solution and the standard test substance solution, respectively placing the sample in 20ul at room temperature for 18 hours, respectively placing the sample on a same silica gel Gs aluminum plate in a strip shape, placing the sample in a chromatographic cylinder with developing agent wetting filter paper attached to the inner wall, developing and airing. Sprayed with a copper sulphate phosphoric acid solution and heated at 170 ℃ for 10 minutes. And observing the spot colors of the reference substance solution, the test substance solution and the labeled test substance solution. The results are shown in the following table and the typical pattern is shown in FIG. 9.
TABLE 7 DSPG free fatty acid detection results of Na
Comparative example 2 literature method
Reference "determination of phospholipid content in liposomes by HPLC-electrospray detector (CAD) assay" derived from "journal of Chinese pharmacy", volume 42, 23, 12 months of 2007
Instrument Siemens flight U3000 equipped with electrospray detector
Chromatographic conditions:
The chromatographic column is Thermo SCIENTIFIC HYPERSIL GOLD C18.6X250 mm,5um, water as mobile phase A, methanol as mobile phase B, CAD atomization temperature 35 ℃, column temperature 30 ℃, flow rate 1ml/min, sample injection volume 10ul, and gradient elution according to the following table:
Table 8 elution gradient table in comparative example 2
The solution is prepared by weighing about 24mg of DSPG-Na in a 20ml measuring flask, dissolving in solvent, diluting to scale, shaking, taking 10ul, and loading into chromatograph, and recording chromatogram. The result is shown in FIG. 10, where DSPG-Na peaks were trailing.
The following comparative examples 3-5 are high performance liquid chromatography combined with electrospray detector (HPLC-CAD) methods using other conditions, these comparative examples are not exhaustive, and the application finally obtains the analytical detection method of the related substances of distearoyl phosphatidyl sodium by searching and researching various test conditions and parameters, and the established methods of example 1, example 4 and example 5 are the most suitable analytical detection methods of the related substances of distearoyl phosphatidyl sodium.
Comparative example 3
Instrument sameifeu 3000 is equipped with an electrospray detector.
Chromatographic conditions:
Octyl silane bonded silica gel column (Luna cube 5 mu m C8 (2) 100A, 250X 4.6 mm), 0.1mol/L ammonium acetate solution (pH value adjusted to 3.0 by acetic acid) methanol (75:25) mobile phase A, methanol as mobile phase B, flow rate of 1.0ml/min, CAD detector atomization temperature of 35 ℃, column temperature of 30 ℃ and sample injection volume of 50 μl. The sample pan temperature was 25℃and elution was carried out gradient according to the table below.
TABLE 9 elution gradient mode for comparative example 3
Preparation of mixed solution [ adding formulation prescription ingredients, namely excipient and dipalmitoyl phosphatidylcholine (DPPC) and related impurities Lysophosphatidylcholine (LPPC) and palmitic acid ] based on DSPG-Na and related impurities ]:
Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoyl phosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC solubilizer [ water-t-butanol (1:1) ] were dissolved and quantitatively diluted to prepare a mixed solution containing 14mg mannitol, about 25 μg LPPC, about 36 μg LSPG-Na, about 16 μg palmitic acid, about 36 μg stearic acid, about 36 μg phosphatidic acid, and about 36 μg phosphatidylcholine per 1ml DPPC, about 0.8mg DSPG-Na, and about 3.6mg DSPG-Na. And (5) sample injection. The results are shown in FIG. 11. Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoyl phosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC sequentially show peaks, and DSPA-Na peaks and DSPC peaks coincide without separation.
Comparative example 4
Instrument sameifeu 3000 is equipped with an electrospray detector.
Chromatographic conditions:
Octyl silane bonded silica gel column (Luna 5. Mu. m C8 (2) 100A, 250X 4.6 mm), 0.1mol/L ammonium acetate solution (pH adjusted to 3.0 with acetic acid) methanol (75:25) mobile phase A, 0.1mol/L ammonium acetate solution (pH adjusted to 3.0 with acetic acid) methanol (5:95) mobile phase B, flow rate of 1.0ml/min, CAD detector atomization temperature of 35 ℃, column temperature of 30 ℃ and sample injection volume of 50. Mu.l. The sample pan temperature was 25℃and elution was carried out gradient according to the table below.
TABLE 10 elution gradient mode for comparative example 4
Preparing a mixed solution:
Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoyl phosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC solubilizer [ water-t-butanol (1:1) ] were dissolved and quantitatively diluted to prepare a mixed solution containing 14mg mannitol, about 25 μg LPPC, about 36 μg LSPG-Na, about 16 μg palmitic acid, about 36 μg stearic acid, about 36 μg phosphatidic acid, and about 36 μg phosphatidylcholine per 1ml DPPC, about 0.8mg DSPG-Na, and about 3.6mg DSPG-Na. And (5) sample injection. The results are shown in FIG. 12. Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoyl phosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC sequentially peak, and the peak separation between DSPA-Na and DSPC peaks is not satisfactory. After the mixed solution DSPG-Na, there are only 2 impurity peaks, DSPA-Na and DSPC, respectively, and the unknown impurities of DSPG-Na are not eluted.
Comparative example 5
Instrument sameifeu 3000 is equipped with an electrospray detector.
Chromatographic conditions:
Octyl silane bonded silica gel column (Luna 5. Mu. m C8 (2) 100A, 250X 4.6 mm), 0.1mol/L ammonium acetate solution (pH adjusted to 3.0 with acetic acid) methanol (75:25) mobile phase A, 0.1mol/L ammonium acetate solution (pH adjusted to 3.0 with acetic acid) methanol (5:95) mobile phase B, flow rate of 1.0ml/min, CAD detector atomization temperature of 35 ℃, column temperature of 30 ℃ and sample injection volume of 50. Mu.l. The sample pan temperature was 25℃and elution was carried out gradient according to the table below.
TABLE 11 elution gradient mode of comparative example 5
Preparing a mixed solution:
Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoyl phosphatidylcholine (DPPC), DSPG-Na, DSPA-Na, and DSPC solubilizer [ water-t-butanol (1:1) ] were dissolved and quantitatively diluted to prepare a mixed solution containing 14mg mannitol, about 25 μg LPPC, about 36 μg LSPG-Na, about 16 μg palmitic acid, about 36 μg stearic acid, about 36 μg phosphatidic acid, and about 36 μg phosphatidylcholine per 1ml DPPC, about 0.8mg DSPG-Na, and about 3.6mg DSPG-Na. And (5) sample injection. The results are shown in FIG. 13. Mannitol (excipient), lysophosphatidylcholine (LPPC), LSPG-Na, palmitic acid, stearic acid, dipalmitoyl phosphatidylcholine (DPPC), DSPG-Na, DSPA-Na and DSPC sequentially show peaks, the peak separation degree between DSPA-Na peaks and DSPC peaks is not satisfactory, but after DSPG-Na, 3 impurity peaks exist, and unknown impurities of DSPG-Na can be eluted.
The method has few high performance liquid chromatography methods for analysis and detection of distearoyl phosphatidyl sodium related substances, and only a very small number of published schemes exist at present, such as a literature method for determining the content of phospholipid in liposome by using an HPLC-electrospray detector (CAD) detection method in comparative example 2, but peak tailing and poor determination effect are caused, so that the conditions of the high performance liquid chromatography method for analysis and detection of distearoyl phosphatidyl sodium related substances are difficult to obtain, and the method is proved to be also capable of confirming the problem through fumbling research of various test conditions and parameters.
Through a great deal of research work, the invention develops a detection method aiming at distearoyl phosphatidyl sodium glycerate and various related substances thereof, and solves various problems in detecting the related substances of the substances. The analysis and detection method can simultaneously separate and detect distearoyl phosphatidyl glycerol sodium and impurities I, II, III, IV and IV, wherein the impurities I and II are lysophosphatidyl glycerol, stearic acid and phosphatidic acid.
As can be seen from the test results of the examples and the comparative examples, the detection and analysis method provided by the invention is more visual, and 4 known impurities and other unknown impurities in the sample can be detected by using one chromatographic condition.
And through method verification, the method has the advantages that the detection and control of DSPG-Na related substances can be better and more conveniently realized through the requirements of method verification on the aspects of specificity, detection limit and quantitative limit, linearity, accuracy, durability and the like.
The present invention is not limited to the preferred embodiments, and any simple modification, equivalent replacement, and improvement made to the above embodiments by those skilled in the art without departing from the technical scope of the present invention, will fall within the scope of the present invention.
Claims (10)
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