Method for detecting phospholipids and impurities thereof

Abstract

The invention relates to a method for detecting phospholipid and impurities thereof, which comprises the following steps: dissolving and / or diluting a sample to be detected in a diluent to prepare a test solution; performing high performance liquid chromatography detection on the test solution; wherein the conditions of the high performance liquid chromatography detection are as follows: (1) a filling material of a chromatographic column is polyvinyl alcohol bonded silica gel; (2) a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises normal hexane and isopropanol, and the mobile phase B comprises isopropanol and water; gradient elution is adopted. The detection method has the advantages of multiple types of separated phospholipids and impurities, high separation degree, short time consumption and low cost.

Description

Technical Field

[0001] This application relates to the technical field of chromatographic analysis, and particularly to a method for detecting phospholipids and their impurities. Background Art

[0002] Phospholipids are lipid compounds containing phosphoric acid, belonging to a type of complex lipid and being the main component of biological membranes. Phospholipids are present in almost all body cells, and there are relatively large amounts of phospholipids in important tissues of animals and plants, such as egg yolks, milk, the brain tissue, liver, kidneys, and muscle tissues of animals, or oil seeds, etc.

[0003] During the processing of products, phospholipid impurities will remain in the products, thereby affecting the quality and function of the products. For example, the phospholipid impurities remaining in microspheres with phospholipids as the main excipients in contrast agents will affect their imaging effect, and thus affect medical judgment. The effective monitoring and control of phospholipid impurities can directly affect the safety and effectiveness of the finished preparation. However, phospholipid molecules are a type of amphoteric compound with phosphate groups, containing both a hydrophobic tail composed of fatty acid chains (Hydropholilictail) and a hydrophilic head composed of a substituted group connected by phosphoric acid (Hydrophilichead). Each phospholipid is not a simple compound. According to the polar head group, the different carbon chain lengths, and the number of unsaturated bonds, the composition of phospholipids is highly complex and shows diverse characteristics. Therefore, the analysis of phospholipids is quite difficult. Therefore, there are many types of phospholipids and their impurities in products, and phospholipids mainly include sodium distearoyl phosphatidate (DSPA), sodium distearoyl phosphatidylserine (DSPS), and phospholipid impurities include distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine (DSPE), sodium distearoyl phosphatidylglycerol (DSPG), sodium lysophosphatidic acid (Lyso-PA, LPA), sodium lysophosphatidylserine (Lyso-PS, LPS), etc.

[0004] To better monitor the phospholipids and their impurities in products, the literature method "Quantification of phosphatidylserine, phosphatidic acid and free fatty acids in an ultrasound contrast agent by normal-phase high-performance liquid chromatography with evaporative light scattering detection" uses a pre-column Merck LiChrospher 100 Diol and a chromatographic column Merck LiChroCART, LiChrospher 100 Diol for liquid chromatography detection. However, this method can separate fewer phospholipids and their impurities, some phospholipid impurities (such as Lyso-PS and Lyso-PA) cannot achieve baseline separation, the analysis takes a long time, and the separation mode of the pre-column plus chromatographic column also increases the analysis cost. Summary of the Invention

[0005] Based on this, the present application provides a detection method for phospholipids and their impurities with a large variety of separated phospholipids and their impurities, high resolution, short time consumption, and low cost.

[0006] The specific technical solution is as follows:

[0007] A detection method for phospholipids and their impurities, comprising the following steps:

[0008] Dissolve and / or dilute the sample to be tested in a diluent to prepare a test solution;

[0009] Perform high-performance liquid chromatography detection on the test solution;

[0010] Among them, the conditions for the high-performance liquid chromatography detection include:

[0011] (1) The packing material of the chromatographic column is polyvinyl alcohol-bonded silica gel;

[0012] (2) The mobile phase includes mobile phase A and mobile phase B. Mobile phase A includes n-hexane and isopropanol, and mobile phase B includes isopropanol and water; gradient elution is used.

[0013] In some embodiments, the elution program of the gradient elution includes:

[0014] 0 - 3.5 min, the volume percentage of mobile phase A is maintained at 82%, and the volume percentage of mobile phase B is maintained at 18%;

[0015] From 3.5 min to 11 min, the volume percentage of mobile phase A changes from 82% to 20% - 30%, and the volume percentage of mobile phase B changes from 18% to 80% - 70%.

[0016] From 11 min to 12 min, the volume percentage of mobile phase A remains at 20% - 30%, and the volume percentage of mobile phase B remains at 80% - 70%.

[0017] From 12 min to 12.1 min, the volume percentage of mobile phase A changes from 20% - 30% to 82%, and the volume percentage of mobile phase B changes from 80% - 70% to 18%.

[0018] From 12.1 min to 18 min, the volume percentage of mobile phase A remains at 82%, and the volume percentage of mobile phase B remains at 18%.

[0019] In some embodiments, the elution program of the gradient elution includes:

[0020] From 0 to 3.5 min, the volume percentage of mobile phase A remains at 82%, and the volume percentage of mobile phase B remains at 18%.

[0021] From 3.5 min to 11 min, the volume percentage of mobile phase A changes from 82% to 25%, and the volume percentage of mobile phase B changes from 18% to 75%.

[0022] From 11 min to 12 min, the volume percentage of mobile phase A remains at 25%, and the volume percentage of mobile phase B remains at 75%.

[0023] From 12 min to 12.1 min, the volume percentage of mobile phase A changes from 25% to 82%, and the volume percentage of mobile phase B changes from 75% to 18%.

[0024] From 12.1 min to 18 min, the volume percentage of mobile phase A remains at 82%, and the volume percentage of mobile phase B remains at 18%.

[0025] In some embodiments, mobile phase A includes n - hexane, isopropanol, acetic acid, and triethylamine with a volume ratio of (830 - 850):(140 - 160):(0 - 24):(0 - 3); and / or, mobile phase B includes isopropanol, water, acetic acid, and triethylamine with a volume ratio of (810 - 830):(170 - 190):(0 - 24):(0 - 3).

[0026] In some of these embodiments, mobile phase A comprises n-hexane, isopropanol, acetic acid, and triethylamine in a volume ratio of (830 to 850):(140 to 160):(8 to 12):(0.5 to 1.5); and / or, mobile phase B comprises isopropanol, water, acetic acid, and triethylamine in a volume ratio of (810 to 830):(170 to 190):(8 to 12):(0.5 to 1.5).

[0027] In some of these embodiments, the packing particle size of the chromatographic column is 3 μm to 5 μm, the length of the chromatographic column is 150 mm to 250 mm, and the diameter of the chromatographic column is 2 mm to 5 mm.

[0028] In some of these embodiments, the diluent is chloroform, methanol, and water in a volume ratio of (60 to 90):(10 to 40):(0 to 4).

[0029] In some of these embodiments, the conditions for the high performance liquid chromatography detection further include one or more of the following features:

[0030] (1) The injection volume is 5 μL to 25 μL;

[0031] (2) The flow rate is 0.9 to 1.1 mL / min;

[0032] (3) The column temperature is 35°C to 45°C;

[0033] (4) The detector is an evaporative light scattering detector or an electrospray detector;

[0034] Optionally, the drift tube temperature of the evaporative light scattering detector is 100°C to 110°C, and the carrier gas flow rate is 1.7 to 1.9 L / min;

[0035] Optionally, the nebulizer temperature of the electrospray detector is 35°C to 50°C, the power rate is 1.0 to 1.3, the sampling frequency is 5 to 15 Hz, and the filter constant is 3.0 to 7.0 s.

[0036] In some of these embodiments, the phospholipids and their impurities include one or more of sodium distearoylphosphate, sodium distearoylphosphatidylserine, distearoylphosphatidylcholine, distearoylethanolamine, sodium distearoylphosphatidylglycerol, sodium lysophosphatide, and sodium lysophosphatidylserine.

[0037] In some of these embodiments, the method for detecting the phospholipids and their impurities further includes the following steps:

[0038] Dissolve and / or dilute the reference substance of the phospholipids and their impurities in the diluent to prepare reference substance solutions with different concentrations;

[0039] Perform high - performance liquid chromatography (HPLC) detection on the reference substance solutions with different concentrations, and construct a standard curve for phospholipids and their impurities based on the detection results.

[0040] Substitute the detection results of the HPLC detection of the test sample into the standard curve of the phospholipids and their impurities to obtain the contents of the phospholipids and their impurities in the test sample.

[0041] The above - mentioned detection method for phospholipids and their impurities is based on high - performance liquid chromatography. By reasonably setting the chromatographic column packing and the mobile phase, it can achieve the separation of various phospholipids and their impurities in the test sample. Moreover, this method has a short elution time, high detection sensitivity, strong specificity, high precision, high accuracy, and convenient operation. While improving the detection timeliness, convenience, and universality, it can also reduce the usage amount of highly toxic reagents, being more energy - saving and environmentally friendly. Specifically, compared with the traditional method, the beneficial effects of the above - mentioned detection method for phospholipids and their impurities are also reflected in:

[0042] (1) It does not require the use of a pre - column, is more economical, and reduces the analysis cost.

[0043] (2) It has a shorter elution time and can separate more types of phospholipids and their impurities.

[0044] (3) It solves the deficiency that some components, such as Lyso - PS and Lyso - PA, are not baseline - separated.

[0045] (4) The elution solvent is less toxic and more environmentally friendly, reducing the analysis risk and cost. Description of the Drawings

[0046] Figure 1 It is the chromatogram of the system suitability solution in Example 1. Among them, Peak 1: solvent peak (RT: 1 min - 2 min), Peak 2: DSPE, Peak 3: DSPA, Peak 4: DSPC, Peak 5: DSPG, Peak 6: DSPS, Peak 7: lyso - PA, Peak 8: lyso - PS, Peak 9: solvent peak (RT: 11 min - 14.5 min).

[0047] Figure 2 It is the chromatogram of the system suitability solution 2 in Example 6. Among them, Peak 1: solvent peak (RT: 1 min - 2 min), Peak 2: DSPE, Peak 3: DSPA, Peak 4: DSPC, Peak 5: DSPS, Peak 6: lyso - PA, Peak 7: lyso - PS, Peak 8: solvent peak (RT: 11 min - 14.5 min).

[0048] Figure 3It is the chromatogram of the system suitability solution 3 in Example 6, where Peak 1: solvent peak (RT: 1 min - 2 min), Peak 2: DSPE, Peak 3: DSPA, Peak 4: DSPC, Peak 5: DSPS, Peak 6: lyso-PA, Peak 7: lyso-PS, Peak 8: solvent peak (RT: 11 min - 14.5 min). Detailed implementation manners

[0049] The following further elaborates on the method for detecting phospholipids and their impurities in this application in combination with specific examples. This application can be implemented in many different forms and is not limited to the implementation manners described herein. On the contrary, the purpose of providing these implementation manners is to make the understanding of the disclosed content of this application more thorough and comprehensive.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific examples and are not intended to limit this application.

[0051] The optional ranges of the terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items.

[0052] In this article, "one or more" refers to any one, any two, or any two or more of the listed items.

[0053] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, as well as an open technical solution containing the listed features.

[0054] In this application, regarding the numerical range, unless otherwise specified, the above numerical range is considered continuous and includes the minimum value and the maximum value of this range, as well as each value between such minimum value and maximum value. Further, when the range refers to an integer, it includes each integer between the minimum value and the maximum value of this range. In addition, when providing multiple ranges to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0055] The percentage content involved in this application, unless otherwise specified, refers to the mass percentage for solid-liquid mixing and solid-solid mixing, and refers to the volume percentage for liquid-liquid mixing.

[0056] In this application, the percentage concentration, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0057] In this application, the temperature parameter, unless otherwise specified, allows for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0058] Room temperature in this application generally refers to 4°C to 30°C, preferably 20 ± 5°C.

[0059] Some examples of this application provide a method for detecting phospholipids and their impurities, including the following steps:

[0060] Dissolve and / or dilute the sample to be tested in a diluent to prepare a test solution;

[0061] Perform high performance liquid chromatography (HPLC) detection on the test solution;

[0062] Among them, the conditions for the HPLC detection include:

[0063] (1) The packing material of the chromatographic column is polyvinyl alcohol bonded silica gel;

[0064] (2) The mobile phase includes mobile phase A and mobile phase B. Mobile phase A includes n-hexane and isopropanol, and mobile phase B includes isopropanol and water; gradient elution is adopted.

[0065] In some of these examples, the elution program for the gradient elution includes:

[0066] 0 - 3.5 min, the volume percentage of mobile phase A remains 82%, and the volume percentage of mobile phase B remains 18%;

[0067] 3.5 min - 11 min, the volume percentage of mobile phase A changes from 82% to 20% - 30%, and the volume percentage of mobile phase B changes from 18% to 80% - 70%; specifically, the percentage that the volume percentage of mobile phase A changes from 82% to includes, but is not limited to: 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or the range between any two of the foregoing; the volume percentage of mobile phase B changes accordingly;

[0068] 11 min - 12 min, the volume percentage of mobile phase A remains 20% - 30%, and the volume percentage of mobile phase B remains 80% - 70%;

[0069] From 12 min to 12.1 min, the volume percentage of mobile phase A changes from 20% - 30% to 82%, and the volume percentage of mobile phase B changes from 80% - 70% to 18%.

[0070] From 12.1 min to 18 min, the volume percentage of mobile phase A remains at 82%, and the volume percentage of mobile phase B remains at 18%.

[0071] Understandably, the total volume percentage of mobile phase A and mobile phase B is 100%.

[0072] In some of these examples, the elution program of the gradient elution includes:

[0073] From 0 to 3.5 min, the volume percentage of mobile phase A remains at 82%, and the volume percentage of mobile phase B remains at 18%.

[0074] From 3.5 min to 11 min, the volume percentage of mobile phase A changes from 82% to 25%, and the volume percentage of mobile phase B changes from 18% to 75%.

[0075] From 11 min to 12 min, the volume percentage of mobile phase A remains at 25%, and the volume percentage of mobile phase B remains at 75%.

[0076] From 12 min to 12.1 min, the volume percentage of mobile phase A changes from 25% to 82%, and the volume percentage of mobile phase B changes from 75% to 18%.

[0077] From 12.1 min to 18 min, the volume percentage of mobile phase A remains at 82%, and the volume percentage of mobile phase B remains at 18%.

[0078] In some of these examples, mobile phase A includes n - hexane, isopropanol, acetic acid, and triethylamine with a volume ratio of (830 - 850):(140 - 160):(0 - 24):(0 - 3). Further, mobile phase A includes n - hexane, isopropanol, acetic acid, and triethylamine with a volume ratio of (830 - 850):(140 - 160):(8 - 12):(0.5 - 1.5). Even further, mobile phase A includes n - hexane, isopropanol, acetic acid, and triethylamine with a volume ratio of 840:150:10:0.8.

[0079] In some of these examples, mobile phase B comprises isopropanol, water, acetic acid, and triethylamine in a volume ratio of (810 - 830):(170 - 190):(0 - 24):(0 - 3). Further, mobile phase B comprises isopropanol, water, acetic acid, and triethylamine in a volume ratio of (810 - 830):(170 - 190):(8 - 12):(0.5 - 1.5). Still further, mobile phase B comprises isopropanol, water, acetic acid, and triethylamine in a volume ratio of 820:180:10:0.8.

[0080] In some of these examples, the packing particle size of the chromatographic column is 3 μm - 5 μm, the length of the chromatographic column is 150 mm - 250 mm, and the diameter of the chromatographic column is 2 mm - 5 mm. Further, the packing particle size of the chromatographic column is 5 μm, the length of the chromatographic column is 250 mm, and the diameter of the chromatographic column is 4.6 mm. Still further, the model of the chromatographic column is YMC - Pack PVA - SIL - NP.

[0081] In some of these examples, the diluent is chloroform, methanol, and water in a volume ratio of (60 - 90):(10 - 40):(0 - 4). Specifically, the volume ratio of chloroform, methanol, and water includes but is not limited to: 90:10:0, 70:20:4, 65:25:4, 60:30:4, 60:40:0, or the range between any two of the foregoing.

[0082] In some of these examples, the conditions for high - performance liquid chromatography detection further include one or more of the following features:

[0083] (1) The injection volume is 5 μL - 25 μL; specifically, the injection volume includes but is not limited to: 5 μL, 7 μL, 10 μL, 13 μL, 15 μL, 17 μL, 20 μL, 22 μL, 25 μL, or the range between any two of the foregoing;

[0084] (2) The flow rate is 0.9 - 1.1 mL / min; specifically, the flow rate includes but is not limited to: 0.9 mL / min, 1 mL / min, 1.1 mL / min;

[0085] (3) The column temperature is 35°C - 45°C; specifically, the column temperature includes but is not limited to: 35°C, 38°C, 40°C, 42°C, 45°C, or the range between any two of the foregoing;

[0086] (4) The detector is an evaporative light - scattering detector (ELSD) or an charged aerosol detector (CAD).

[0087] Without limitation, the drift tube temperature of the evaporative light scattering detector is 100°C to 110°C, and the carrier gas flow rate is 1.7 to 1.9 L / min. Specifically, the drift tube temperature includes but is not limited to: 100°C, 103°C, 105°C, 107°C, 110°C, or the range between any two of the foregoing; the carrier gas flow rate includes but is not limited to: 1.7 L / min, 1.8 L / min, 1.9 L / min, or the range between any two of the foregoing.

[0088] Without limitation, the nebulizer temperature of the electrospray detector is 35°C to 50°C, the power rate is 1.0 to 1.3, the sampling frequency is 5 Hz to 15 Hz, and the filter constant is 3.0 s to 7.0 s. Specifically, the nebulizer temperature includes but is not limited to: 35°C, 40°C, 45°C, 50°C, or the range between any two of the foregoing. Specifically, the power rate includes but is not limited to: 1.0, 1.1, 1.15, 1.2, 1.25, 1.3, or the range between any two of the foregoing. Specifically, the sampling frequency includes but is not limited to: 5 Hz, 8 Hz, 10 Hz, 12 Hz, 15 Hz, or the range between any two of the foregoing. Specifically, the filter constant includes but is not limited to: 3.0 s, 4.0 s, 5.0 s, 6.0 s, 7.0 s, or the range between any two of the foregoing.

[0089] In some of these examples, the phospholipids and their impurities include one or more of sodium distearoyl phosphatidate (DSPA), sodium distearoyl phosphatidylserine (DSPS), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine (DSPE), sodium distearoyl phosphatidylglycerol (DSPG), lysophosphatidic acid sodium salt (Lyso-PA, LPA), and lysophosphatidylserine sodium salt (Lyso-PS, LPS).

[0090] In some of these examples, the method for detecting the phospholipids and their impurities further includes the following steps: dissolving and / or diluting the reference substances of the phospholipids and their impurities in a diluent to prepare reference substance solutions with different concentrations; performing high performance liquid chromatography detection on the reference substance solutions with different concentrations, and constructing a standard curve for the phospholipids and their impurities according to the detection results;

[0091] Substituting the detection results of the high performance liquid chromatography detection of the test sample into the standard curve of the phospholipids and their impurities to obtain the content of the phospholipids and their impurities in the test sample.

[0092] Additionally without limitation, the test sample can be a phospholipid-containing preparation, such as perfluorobutane microspheres for injection, perfluoropropane microspheres, sulfur hexafluoride microbubbles, or phospholipid extracts of the above preparations.

[0093] For the experimental parameters not specified in the following specific embodiments, priority shall be given to referring to the guidance provided in this application document. It is also possible to refer to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by the manufacturer.

[0094] The raw materials and reagents involved in the following specific embodiments can be obtained commercially, or can be prepared by those skilled in the art according to known means.

[0095] Example 1

[0096] This example constructs a detection method for phospholipids and their impurities.

[0097] High-performance liquid chromatograph: Agilent 1260;

[0098] Chromatographic column: YMC-Pack PVA-SIL-NP, 4.6×250mm, 5μm;

[0099] Flow rate: 1.0 ml / min;

[0100] Column temperature: 40°C;

[0101] The detector is an evaporative light scattering detector, drift tube temperature: 105°C, carrier gas flow rate: 1.8 L / min;

[0102] Injection volume: 15 μL;

[0103] Mobile phase A: n-hexane - isopropanol - acetic acid - triethylamine (840:150:10:0.8, v:v:v:v);

[0104] Mobile phase B: isopropanol - water - acetic acid - triethylamine (820:180:10:0.8, v:v:v:v);

[0105] The elution program is shown in Table 1 below (volume percentage):

[0106] Table 1

[0107]

[0108] Diluent: chloroform - methanol - water (65:25:4, v:v:v);

[0109] Reference solution: Accurately weigh about 1.0 mg of impurity reference standards LPA, LPS, DSPC, DSPE, and DSPG respectively, place them in 5-mL volumetric flasks, add diluent, sonicate to dissolve, and dilute to the mark. Shake well to obtain single-impurity standard solutions; pipette 0.7 mL of the LPA single-impurity standard solution, and about 1.4 mL of the single-impurity standard solutions of LPS, DSPC, DSPE, and DSPG into the same 10-mL volumetric flask, dilute to the mark with diluent, and shake well to obtain a mixed-impurity solution; pipette 0.3, 0.45, 0.6, 0.75, and 0.9 mL of the mixed-impurity solution into sample vials respectively, add 0.9, 0.75, 0.6, 0.45, and 0.3 mL of diluent respectively, mix well to obtain the solutions.

[0110] System suitability solution: Weigh about 5 mg of DSPS and about 1 mg of DSPA, place them in a 10-mL volumetric flask, pipette 0.7 mL of the LPA single-impurity standard solution, and about 1.4 mL of the single-impurity standard solutions of LPS, DSPC, DSPE, and DSPG, add diluent, sonicate to dissolve, and dilute to the mark. Shake well to obtain the solution.

[0111] Sample detection: Pipette 15 μL of the system suitability solution accurately, inject it into the liquid chromatograph, and record the chromatogram; the chromatogram of the system suitability solution is as Figure 1 shown.

[0112] From the test results, it can be seen that the separation between sodium distearoyl phosphatidate, sodium distearoyl phosphatidylserine and each impurity is good, and the minimum resolution between impurities can reach more than 1.50, meeting the requirements for impurity separation detection.

[0113] Example 2

[0114] This example is for the construction of a content detection method for phospholipids and their impurities and a recovery test.

[0115] Prepare the reference solution according to Example 1, and detect the reference according to the chromatographic method in Example 1. Construct the linear equations of LPA, LPS, DSPC, DSPE, and DSPG respectively. The results are as follows:

[0116] DSPE: y = 2.0292x - 0.1513, r = 0.998;

[0117] DSPC: y = 2.0593x + 0.1140, r = 0.996;

[0118] DSPG: y = 2.0413x - 0.6947, r = 0.995;

[0119] LPA: y = 1.7323x + 0.0812, r = 0.997;

[0120] LPS: y = 1.5629x + 0.5851, r = 1.000;

[0121] Wherein, x is the logarithm of the impurity concentration, and y is the logarithm of the peak area. It can be seen that the linear correlation coefficients of each linear equation are not less than 0.995.

[0122] Prepare the impurity recovery solution: Take 0.3, 0.6, and 0.6 mL of the system suitability solution prepared in Example 1 and place them into injection vials respectively, add 0.6, 0.6, and 0.3 mL of diluent respectively, and mix well to obtain.

[0123] Detect the impurity recovery solution according to the chromatographic method of Example 1 to obtain the recovery rate, and the results are shown in Table 2 below:

[0124] Table 2

[0125] Impurity Average recovery rate % RSD % DSPE 115.71 2.69 DSPC 123.38 4.99 DSPG 99.15 13.94 LPA 130.68 3.29 LPS 108.02 2.64

[0126] Example 3

[0127] This example is a reproducibility test of the content detection method for phospholipids and their impurities.

[0128] According to the method of Example 1, another operator re-prepared the reference substance and the impurity recovery solution, and detected them on another high-performance liquid chromatograph. The measured linear equation, linear correlation coefficient, and impurity recovery rate had little difference, and the method had good reproducibility.

[0129] Example 4

[0130] This example is an investigation of the elution program of the detection method for phospholipids and their impurities.

[0131] Perform the detection of the system suitability solution in the same manner as in Example 1, and only change the elution program as shown in Table 3 or Table 4 below:

[0132] Table 3

[0133]

[0134] Table 4

[0135]

[0136] From the detection results, it can be seen that when the elution program is as shown in Table 1, the minimum separation degree between impurities is 1.82, which is the best, and the peak shapes of each impurity are good, which is the best. When the elution program is as shown in Table 2, the minimum separation degree between impurities is 1.78. When the elution program is as shown in Table 3, the minimum separation degree between impurities is 1.60.

[0137] Example 5

[0138] This example is for the investigation of diluents in the detection method of phospholipids and their impurities.

[0139] Perform the detection of the system suitability solution as in Example 1, only changing the diluents to Diluent 2: chloroform: methanol = 90:10 (v:v), Diluent 3: chloroform: methanol = 60:40 (v:v), and Diluent 4: mobile phase B, and prepare System Suitability Solution 2, System Suitability Solution 3, and System Suitability Solution 4 respectively.

[0140] Among them, during the preparation of System Suitability Solution 4, Diluent 4 cannot dissolve the sample. Detect System Suitability Solution 2 and System Suitability Solution 3 as in Example 1, and the results are as Figure 2 and Figure 3 shown. It can be seen from the detection results that the proportion of chloroform in Diluent 2 is relatively high, and the peak shape of DSPA collapses significantly. The proportion of methanol in Diluent 3 is relatively high, and the peak of DSPS splits. When the diluent is the diluent of Example 1, the peak shapes of each impurity are sharp, and the impurity response is good, which is the best.

[0141] Example 6

[0142] This example is for the investigation of the injection volume in the detection method of phospholipids and their impurities.

[0143] Perform the detection of the system suitability solution as in Example 1, only changing the injection volumes to 10 μL and 20 μL respectively. It can be seen from the detection results that when the injection volume is 20 μL, the resolution between impurities becomes smaller. When the injection volume is 10 μL, the separation of impurities is higher, but the impurity response decreases. When the injection volume is 15 μL, the minimum resolution between impurities can reach more than 1.50, and the impurity response is higher, which is the best.

[0144] The technical features of the above-described examples can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as within the scope described in this specification.

[0145] The above-described examples only represent several implementation manners of this application, which are convenient for understanding the technical solutions of this application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of this application. It should be noted that for those of ordinary skill in the art, without departing from the concept of this application, several modifications and improvements can still be made, and these all belong to the protection scope of this application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided in this application are all within the protection scope of the appended claims of this application. Therefore, the protection scope of this application patent should be subject to the content of the appended claims, and the specification can be used to explain the content of the claims.

Claims

1. A method for detecting phospholipids and their impurities, characterized in that It includes the following steps: Dissolve and / or dilute the sample to be tested in a diluent to prepare a test solution; Perform high performance liquid chromatography (HPLC) detection on the test solution; Among them, the conditions for the HPLC detection include: (1) The packing material of the chromatographic column is polyvinyl alcohol bonded silica gel; (2) The mobile phase includes mobile phase A and mobile phase B. Mobile phase A includes n-hexane and isopropanol, and mobile phase B includes isopropanol and water; gradient elution is adopted.

2. The detection method of phospholipids and their impurities according to claim 1, characterized in that, The elution program of the gradient elution includes: From 0 to 3.5 min, the volume percentage of mobile phase A is maintained at 82%, and the volume percentage of mobile phase B is maintained at 18%; From 3.5 min to 11 min, the volume percentage of mobile phase A changes from 82% to 20% - 30%, and the volume percentage of mobile phase B changes from 18% to 80% - 70%; From 11 min to 12 min, the volume percentage of mobile phase A is maintained at 20% - 30%, and the volume percentage of mobile phase B is maintained at 80% - 70%; From 12 min to 12.1 min, the volume percentage of mobile phase A changes from 20% - 30% to 82%, and the volume percentage of mobile phase B changes from 80% - 70% to 18%; From 12.1 min to 18 min, the volume percentage of mobile phase A is maintained at 82%, and the volume percentage of mobile phase B is maintained at 18%.

3. The method for detecting phospholipids and their impurities according to claim 1 or 2, characterized in that, The elution program of the gradient elution includes: From 0 to 3.5 min, the volume percentage of mobile phase A is maintained at 82%, and the volume percentage of mobile phase B is maintained at 18%; From 3.5 min to 11 min, the volume percentage of mobile phase A changes from 82% to 25%, and the volume percentage of mobile phase B changes from 18% to 75%; From 11 min to 12 min, the volume percentage of mobile phase A is maintained at 25%, and the volume percentage of mobile phase B is maintained at 75%; From 12 min to 12.1 min, the volume percentage of mobile phase A changes from 25% to 82%, and the volume percentage of mobile phase B changes from 75% to 18%; From 12.1 min to 18 min, the volume percentage of mobile phase A is maintained at 82%, and the volume percentage of mobile phase B is maintained at 18%.

4. The method for detecting phospholipids and their impurities according to any one of claims 1-3, characterized in that Mobile phase A includes n-hexane, isopropanol, acetic acid and triethylamine with a volume ratio of (830 - 850):(140 - 160):(0 - 24):(0 - 3); and / or, mobile phase B includes isopropanol, water, acetic acid and triethylamine with a volume ratio of (810 - 830):(170 - 190):(0 - 24):(0 - 3).

5. The method for detecting phospholipids and their impurities according to any one of claims 1-4, characterized in that, Mobile phase A includes n-hexane, isopropanol, acetic acid and triethylamine with a volume ratio of (830 - 850):(140 - 160):(8 - 12):(0.5 - 1.5); and / or, mobile phase B includes isopropanol, water, acetic acid and triethylamine with a volume ratio of (810 - 830):(170 - 190):(8 - 12):(0.5 - 1.5).

6. The method for detecting phospholipids and their impurities according to any one of claims 1-5, characterized in that, The particle size of the packing material of the chromatographic column is 3 μm - 5 μm, the length of the chromatographic column is 150 mm - 250 mm, and the diameter of the chromatographic column is 2 mm - 5 mm.

7. The method for detecting phospholipids and their impurities according to any one of claims 1-6, characterized in that, The diluent is chloroform, methanol and water with a volume ratio of (60-90):(10-40):(0-4).

8. The detection method of phospholipids and their impurities according to any one of claims 1-7, characterized in that, The conditions for the high performance liquid chromatography detection further include one or more of the following characteristics: (1) The injection volume is 5 μL - 25 μL; (2) The flow rate is 0.9 - 1.1 mL / min; (3) The column temperature is 35°C - 45°C; (4) The detector is an evaporative light scattering detector or an electrospray detector; Optionally, the drift tube temperature of the evaporative light scattering detector is 100°C - 110°C, and the carrier gas flow rate is 1.7 - 1.9 L / min; Optionally, the nebulizer temperature of the electrospray detector is 35°C - 50°C, the power rate is 1.0 - 1.3, the sampling frequency is 5 Hz - 15 Hz, and the filter constant is 3.0 s - 7.0 s.

9. The method for detecting phospholipids and their impurities according to any one of claims 1-8, characterized in that, The phospholipids and their impurities include one or more of sodium distearoyl phosphatidate, sodium distearoyl phosphatidylserine, distearoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, sodium distearoyl phosphatidylglycerol, sodium lysophosphatidate and sodium lysophosphatidylserine.

10. The detection method of phospholipids and their impurities according to any one of claims 1 to 9, characterized in that, It further includes the following steps: Dissolve and / or dilute the reference substance of the phospholipids and their impurities in the diluent to prepare reference substance solutions with different concentrations; Conduct high performance liquid chromatography detection on the reference substance solutions with different concentrations, and construct a standard curve of the phospholipids and their impurities according to the detection results; Substitute the detection result of the high performance liquid chromatography detection of the test sample into the standard curve of the phospholipids and their impurities to obtain the content of the phospholipids and their impurities in the test sample.

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