Salt detection method of dipalmitoyl phosphatidyl glycerol and application of salt detection method

Abstract

The invention provides a salt detection method of dipalmitoyl phosphatidyl glycerol and application of the salt detection method, and relates to the technical field of biology, the detection method comprises the following steps: separating the salt of dipalmitoyl phosphatidyl glycerol in a sample by adopting a high performance liquid chromatography; high performance liquid chromatography detection conditions comprise that a silica gel chromatographic column is adopted as a chromatographic column; the mobile phase system comprises a mobile phase B, a mobile phase C and a mobile phase D; the mobile phase A is methanol, the mobile phase B is normal hexane, the mobile phase C is isopropanol, and the mobile phase D contains methanol and a formic acid-triethylamine aqueous solution. According to the detection method, the defect that the salt detection method of dipalmitoyl phosphatidyl glycerol is lacked in the prior art is relieved.

Description

Technical Field

[0001] The invention relates to the field of analytical technology, and in particular to a dipalmitoylphosphatidylglycerol salt detection method and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Liposomes have both a hydrophilic head and a hydrophobic tail, which can specifically deliver drugs to specific tissues and become carriers for targeted drug delivery. Medicinal lipids are naturally derived from soybean lecithin and egg phospholipids. Lecithin is a class of lipid compounds containing phosphorus. Common phospholipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), inositol phospholipids (PI), phosphoglycerol (PG), etc. In natural products, they often appear in the form of mixtures. At present, the separation and purification of natural phospholipids is difficult. The purity of single components obtained from natural phospholipids is always low. Researchers often obtain single-component phospholipids with a certain structure through chemical synthesis. Therefore, compared with natural phospholipids, synthetic phospholipids have the advantages of high purity and strong stability, and are more suitable for use as pharmaceutical excipients.

[0004] Phospholipids often exist in organisms as an important component of cell membranes. Phospholipids have a large family, and there are differences in the physical and chemical properties of phospholipids. For example, phospholipids have good solubility in solvents such as chloroform and acetonitrile, but when the number of atoms in the acyl carbon chain is low, these phospholipids can also be dissolved in solvents with greater polarity. Therefore, different types of single phospholipids need to be studied according to their own properties. As a phospholipid lipid molecule, PG is generated by the dehydration condensation reaction of the hydroxyl group in glycerol with the carboxyl group in octadecanoic acid, octadecenoic acid and phosphorylcholine. PG is commonly found in bacteria. Since extraction from bacteria involves drug safety issues, PG is difficult to separate and purify. In practical applications, PG, as a drug, can treat neonatal respiratory distress syndrome with a lack of pulmonary surfactant and inhibit viral infections in the respiratory tract; as a pharmaceutical excipient, PG also has the functions of surfactant and drug transport. Under the PG category, dipalmitoylphosphatidylglycerol (DPPG) is more common, and this excipient has been marketed in China and added to injections as a pharmaceutical excipient. Dipalmitoylphosphatidylglycerol exists in the form of its sodium salt, namely, sodium dipalmitoylphosphatidylglycerol (DPPG-Na).

[0005] There are not many methods for detecting phospholipid content. The most common method is the egg yolk lecithin (for injection) content determination method in the Chinese Pharmacopoeia, but this method is not applicable to all types of phospholipids, and there are few other current standards. However, the use of DPPG-Na requires purity, and it is urgent to establish a method for detecting DPPG-Na content.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a method for detecting a salt of dipalmitoylphosphatidylglycerol, so as to alleviate the defect of the current lack of a method for detecting a salt of dipalmitoylphosphatidylglycerol.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] In a first aspect, a method for detecting a salt of dipalmitoylphosphatidylglycerol is provided, the method comprising separating the salt of dipalmitoylphosphatidylglycerol in a sample by high performance liquid chromatography;

[0010] The HPLC detection conditions include:

[0011] The chromatographic column adopts a silica gel chromatographic column;

[0012] The mobile phase system includes mobile phase B, mobile phase C and mobile phase D;

[0013] Mobile phase B was n-hexane, mobile phase C was isopropanol, and mobile phase D contained methanol and formic acid-triethylamine aqueous solution.

[0014] In a second aspect, the invention provides the use of the dipalmitoylphosphatidylglycerol salt detection method described in the first aspect in any one of the following (i) to (iii):

[0015] (i) preparing a salt of dipalmitoylphosphatidylglycerol or performing quality control on a salt of dipalmitoylphosphatidylglycerol;

[0016] (ii) preparing phospholipids or performing quality control on phospholipids;

[0017] (iii) Preparation of pharmaceutical excipients or quality control of pharmaceutical excipients.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] In view of the lack of methods for detecting the content of dipalmitoylphosphatidylglycerol salts, the present invention provides a method for detecting dipalmitoylphosphatidylglycerol salts for the first time. The method separates dipalmitoylphosphatidylglycerol salts from other impurities by optimizing the mobile phase, for example, separating dipalmitoylphosphatidylglycerol sodium (DPPG-Na) from dipalmitoylphosphatidic acid (DPPA), lysopalmitoylphosphatidylglycerol sodium (LPPG-Na), and dipalmitoylphosphatidylcholine (DPPC). The detection method of the present invention is subjected to limit investigation and recovery test to obtain a relatively stable and reliable detection method for dipalmitoylphosphatidylglycerol salts. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the chromatogram obtained by detecting DPPG single standard solution using the method in Example 2;

[0022] Figure 2 This is the chromatogram obtained by detecting LPPG single standard solution using the method in Example 2;

[0023] Figure 3 This is the chromatogram obtained by detecting DPPA single standard solution using the method in Example 2;

[0024] Figure 4 This is the chromatogram obtained by detecting the mixed standard solution of DPPG and DPPC using the method of Example 2;

[0025] Figure 5 This is the chromatogram obtained by detecting the mixed standard solution using the method of Example 2;

[0026] Figure 6 The chromatogram obtained by detecting the blank solution according to the method of Example 2;

[0027] Figure 7 This is the chromatogram obtained by detecting the mixed standard solution using the method of Example 3;

[0028] Figure 8 This is the chromatogram obtained by detecting the mixed standard solution using the method of Comparative Example 1;

[0029] Fig. 9 This is the chromatogram obtained by detecting the mixed standard solution using the method of Comparative Example 2;

[0030] Fig.10 This is the chromatogram obtained by detecting the mixed standard solution using the method of Comparative Example 3;

[0031] Fig.11 This is the chromatogram obtained by detecting the blank solution using the method in Comparative Example 3. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In a first aspect, a method for detecting a salt of dipalmitoylphosphatidylglycerol is provided. The method comprises separating the salt of dipalmitoylphosphatidylglycerol in a sample by high performance liquid chromatography.

[0034] The HPLC detection conditions include the following:

[0035] The mobile phase system includes mobile phase B, mobile phase C and mobile phase D.

[0036] Mobile phase B is n-hexane, mobile phase C is isopropanol, and mobile phase D contains methanol and formic acid-triethylamine aqueous solution. Herein, formic acid-triethylamine aqueous solution is an aqueous solution containing formic acid and triethylamine. With n-hexane as mobile phase B, isopropanol as mobile phase C, and methanol and formic acid-triethylamine aqueous solution as mobile phase D, the three-phase combination can alleviate the problem of large baseline fluctuations during the detection process.

[0037] In an optional embodiment, the volume ratio of methanol to formic acid-triethylamine aqueous solution in mobile phase D is (12-14):2, preferably 13:2.

[0038] In an optional embodiment, the pH of the formic acid-triethylamine aqueous solution is 4.8-5.5, and the amount of formic acid and triethylamine in the aqueous solution is limited so that the pH of the aqueous solution reaches 4.8-5.5. Those skilled in the art can adjust the amount of formic acid and triethylamine according to the final pH of the aqueous solution. The pH value of the formic acid-triethylamine aqueous solution between 4.8 and 5.2 makes the separation effect better, and can effectively separate the analyte from its impurity peak, which is beneficial to the detection of the analyte content in the sample.

[0039] In an optional embodiment, the formic acid-triethylamine aqueous solution is prepared as follows: 0.84 mL of formic acid and 3.43 mL of triethylamine are added to 1000 mL of pure water, and the pH is adjusted to between 4.8 and 5.2 using the formic acid aqueous solution to obtain the formic acid-triethylamine aqueous solution. Further, methanol and triethylamine formate water are mixed in a volume ratio of 13:2 to obtain the mobile phase D.

[0040] In an optional embodiment, the high performance liquid chromatography adopts gradient elution, and the gradient elution procedure is as follows, wherein the percentage of each mobile phase is volume percentage:

[0041] 0-1min, mobile phase B 80%→20%, mobile phase C 20%→72%, mobile phase D 0%→8%;

[0042] 1-4 min, mobile phase B 20%, mobile phase C 72%, mobile phase D 8%;

[0043] 4-8 min, mobile phase B 20%→25%, mobile phase C 72%→50%, mobile phase D 8%→25%;

[0044] 8-12 min, mobile phase B 25%, mobile phase C 50%→45%, mobile phase D 25%→30%;

[0045] 12-16 min, mobile phase B 25%, mobile phase C 45%→30%, mobile phase D 30%→45%;

[0046] 16-23 min, mobile phase B 25% → 0%, mobile phase C 30% → 65%, mobile phase D 45% → 35%;

[0047] 23-27 min, mobile phase B 0%, mobile phase C 65%, mobile phase D 35%;

[0048] 27-28 min, mobile phase B 0%, mobile phase C 65%→100%, mobile phase D 35%→0%;

[0049] 28-33 min, mobile phase B 0%, mobile phase C 100%, mobile phase D 0%;

[0050] 33-34 min, mobile phase B 0%→80%, mobile phase C 100%→20%, mobile phase D 0%;

[0051] 34-40 min, mobile phase B 80%, mobile phase C 20%, mobile phase D 0%.

[0052] In the detection method provided in the first aspect, the chromatographic column adopts a silica gel chromatographic column.

[0053] In an optional embodiment, the chromatographic column has a specification of 250×4.6 mm 5 μm.

[0054] In an optional embodiment, the chromatographic column is a Sepax HP-Silica chromatographic column with a specification of 250×4.6 mm 5 μm.

[0055] In an optional embodiment, the column temperature of the chromatographic column is 35-45°C, preferably 40°C.

[0056] In an optional embodiment, the detector is an evaporative light scattering detector (ELSD).

[0057] In an optional embodiment, when the evaporative light scattering detector is used for detection, the drift tube temperature is 80° C. and the gas flow rate is 2.0 L / min.

[0058] In an optional embodiment, the solvent of the test solution is chloroform, methanol and water.

[0059] In an optional embodiment, the solvent of the test solution is chloroform, methanol and water in a volume ratio of 65:35:4.

[0060] In an optional embodiment, the sample includes one or more of a test sample, a standard sample and a quality control sample.

[0061] In an optional embodiment, the concentration of the sample to be tested in the test solution is 0.1-1 mg / mL, preferably 0.3 mg / mL.

[0062] In an optional embodiment, the detection method further comprises obtaining a chromatogram and calculating the content of the salt of dipalmitoylphosphatidylglycerol.

[0063] In an optional embodiment, the content of the dipalmitoylphosphatidylglycerol salt is analyzed and calculated using an external standard method.

[0064] In an optional embodiment, the external standard method is a standard curve method.

[0065] In an optional embodiment, the dipalmitoylphosphatidylglycerol salt is dipalmitoylphosphatidylglycerol sodium;

[0066] In an optional embodiment, the sample further contains one or more of dipalmitoylphosphatidic acid (DPPA), a salt of lysopalmitoylphosphatidylglycerol, and dipalmitoylphosphatidylcholine (DPPC).

[0067] In an alternative embodiment, the salt of lysopalmitoylphosphatidylglycerol comprises a sodium salt (LPPG-Na).

[0068] In an alternative embodiment, the sample comprises synthetic phospholipids.

[0069] In a second aspect, the invention provides the use of the dipalmitoylphosphatidylglycerol salt detection method described in the first aspect in any one of the following (i) to (iii):

[0070] (i) preparing a salt of dipalmitoylphosphatidylglycerol or performing quality control on a salt of dipalmitoylphosphatidylglycerol;

[0071] (ii) preparing phospholipids or performing quality control on phospholipids;

[0072] (iii) Preparation of pharmaceutical excipients or quality control of pharmaceutical excipients.

[0073] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0074] Example 1

[0075] The difficulty in developing the DPPG-Na content method lies in the separation of DPPG-Na from LPPA and LPPG. When the mobile phase changes greatly, it is easy to cause baseline fluctuations. The interference peaks generated by the fluctuations will further interfere with the system. Therefore, this embodiment uses three mobile phases for separation.

[0076] 1. Sample preparation:

[0077] (1) Preparation of test solution: Take 15 mg of DPPG-Na sample, accurately weigh it, place it in a 50 mL volumetric flask, add chloroform: methanol: water (65:35:4) to dissolve and dilute to the scale, shake well, and use it as the test solution.

[0078] (2) Preparation of stock solution:

[0079] (2.1) Preparation of DPPG-Na (sodium dipalmitoylphosphatidylglycerol) stock solution: Take DPPG-Na reference substance, accurately weigh 10 mg into a 10 mL volumetric flask, add chloroform: methanol: water (65:35:4) to dissolve and dilute to the scale, shake well, and the concentration is 1 mg / mL.

[0080] (2.2) Preparation of DPPA (dipalmitoylphosphatidic acid) stock solution: Take DPPA reference substance, accurately weigh 10 mg into a 10 mL volumetric flask, add chloroform: methanol: water (65:35:4) to dissolve and dilute to the scale, shake well, and the concentration is 1 mg / mL.

[0081] (2.3) Preparation of LPPG-Na (lyso-palmitoylphosphatidylglycerol sodium) stock solution: Take LPPG-Na reference substance, accurately weigh 10 mg into a 10 mL volumetric flask, add chloroform: methanol: water (65:35:4) to dissolve and dilute to the scale, shake well, and the concentration is 1 mg / mL.

[0082] (2.4) Preparation of DPPC (dipalmitoylphosphatidylcholine) stock solution: Take DPPC reference substance, accurately weigh 10 mg into a 10 mL volumetric flask, add chloroform: methanol: water (65:35:4) to dissolve and dilute to the scale, shake well, and the concentration is 1 mg / mL.

[0083] (3) Preparation of DPPG-Na standard curve solution: Take DPPG-Na stock solution, accurately take 1 mL into a 2 mL volumetric flask, 0.8 mL into a 2 mL volumetric flask, 0.6 mL into a 2 mL volumetric flask, 0.4 mL into a 2 mL volumetric flask, and 0.2 mL into a 2 mL volumetric flask, and dilute to the scale with chloroform:methanol:water (65:35:4) to obtain DPPG-Na standard curve solution 5-1, with concentrations of 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, and 0.1 mg / mL, respectively.

[0084] (4) Mixed standard solution: Take 1 mL of DPPG-Na stock solution, DPPA stock solution, LPPG-Na stock solution, and DPPC stock solution, and accurately pipette 1 mL each into a graduated test tube. Shake well to obtain a mixed standard solution with a concentration of 0.25 mg / mL for each solution.

[0085] (5) Blank solution: a mixed solution of chloroform: methanol: water in a volume ratio of 65:35:4.

[0086] 2. Preparation of mobile phase:

[0087] Mobile phase B: n-hexane;

[0088] Mobile phase C: isopropanol;

[0089] Mobile phase D: methanol: triethylamine formate water = 13:2 (triethylamine formate pH is 4.8), measure 0.84mL formic acid (concentration 88%), 3.43mL triethylamine, add to 1000mL pure water, use 2mol / L formic acid aqueous solution to adjust the pH to 4.8 to obtain formic acid-triethylamine aqueous solution; mix them in the ratio of methanol: triethylamine formate water = 13:2, and shake well.

[0090] 3. Chromatographic determination and chromatographic conditions:

[0091] Take the blank solution, test solution, mixed standard solution and DPPG-Na standard curve solution prepared in step 1, and inject them into the chromatograph for determination according to the following HPLC conditions. The HPLC conditions are as follows:

[0092] Chromatographic column: Sepax HP-Silica 250×4.6mm 5μm;

[0093] Instrument: Thermo Fisher U3000 with ELSD detector;

[0094] Flow rate: 1.0 mL / min;

[0095] Column oven: 40°C;

[0096] Injection volume: 20 μL;

[0097] ELSD detector: drift tube 80 °C, flow rate 2.0 L / min, split flow, gain 1.

[0098] Gradient elution:

[0099] 0-1min, mobile phase B 80%→20%, mobile phase C 20%→72%, mobile phase D 0%→8%;

[0100] 1-4 min, mobile phase B 20%, mobile phase C 72%, mobile phase 8%;

[0101] 4-8 min, mobile phase B 20%→25%, mobile phase C 72%→50%, mobile phase D 8%→25%;

[0102] 8-12 min, mobile phase B 25%, mobile phase C 50%→45%, mobile phase D 25%→30%;

[0103] 12-16 min, mobile phase B 25%, mobile phase C 45%→30%, mobile phase D 30%→45%;

[0104] 16-23 min, mobile phase B 25% → 0%, mobile phase C 30% → 65%, mobile phase D 45% → 35%;

[0105] 23-27 min, mobile phase B 0%, mobile phase C 65%, mobile phase D 35%;

[0106] 27-28 min, mobile phase B 0%, mobile phase C 65%→100%, mobile phase D 35%→0%;

[0107] 28-33 min, mobile phase B 0%, mobile phase C 100%, mobile phase D 0%;

[0108] 33-34 min, mobile phase B 0%→80%, mobile phase C 100%→20%, mobile phase D 0%;

[0109] 34-40 min, mobile phase B 80%, mobile phase C 20%, mobile phase D 0%.

[0110] Example 2

[0111] 1. Sample preparation:

[0112] The following single standard / mixed standard solutions were prepared using the stock solution prepared in Example 1:

[0113] (1) Add 0.2 mL of DPPG and 0.2 mL of DPPC stock solutions to 1 mL of chloroform: methanol: water (65:35:4) to obtain a DPPG and DPPC mixed standard solution with a concentration of 0.2 mg / mL each.

[0114] (2) Accurately take 0.1 mL of DPPG-Na and add chloroform: methanol: water (65:35:4) to dilute to 1 mL to obtain a single-label DPPG-Na solution with a concentration of 0.1 mg / mL.

[0115] (3) Accurately take 0.1 mL of LPPG and add chloroform: methanol: water (65:35:4) to dilute to 1 mL to obtain a single-label LPPG solution with a concentration of 0.1 mg / mL.

[0116] (4) Accurately take 0.1 mL of DPPA and add chloroform: methanol: water (65:35:4) to dilute to 1 mL to obtain a single-label DPPA solution with a concentration of 0.1 mg / mL.

[0117] 2. Preparation of mobile phase: Same as Example 1.

[0118] 3. Chromatographic determination and chromatographic conditions:

[0119] The chromatographic conditions were the same as in Example 1.

[0120] Take the mixed standard solution and blank solution prepared in step 1 of Example 1, as well as the mixed standard solution of DPPG and DPPC, DPPG-Na single standard, LPPG single standard and DPPA single standard prepared in this example, inject them into the chromatograph for measurement, and record their chromatograms. Figure 1 to Figure 6 The separation degree of each component in the mixed standard solution is shown in Table 1.

[0121] Table 1 Separation of components in the mixed standard solution of Example 1

[0122] name DPPG-Na DPPA LPPG DPPC Separation 2.87 1.85 0.98 /

[0123] Depend on Figure 3 It can be seen that DPPA has a tailing phenomenon in this system, which leads to the stacking phenomenon of LPPG, DPPA, and DPPC in the four control mixed standards. In the mixed standard solution, the separation degree of each component peak and the next identified peak is checked, and it is found that the separation degree of DPPG-Na is 2.89, which is greater than 1.5. It is believed that DPPG-Na can be completely separated in this system. In this system, the baseline of the blank injection at 14 to 19 minutes is relatively flat, and there is no interference peak to interfere with DPPG-Na.

[0124] Example 3

[0125] The pH value of the mobile phase may affect the separation degree. In order to further confirm the stable mobile phase conditions, the pH value was investigated in Example 3.

[0126] 1. Sample preparation:

[0127] Take 0.2 mL of each stock solution in Example 1 and put it in a 1 mL volumetric flask, add chloroform: methanol: water (65:35:4) to dilute to the scale to obtain a mixed standard solution.

[0128] 2. Preparation of mobile phase:

[0129] Mobile phase B: n-hexane;

[0130] Mobile phase C: isopropanol;

[0131] Mobile phase D: methanol: triethylamine formate water = 13:2 (triethylamine formate pH is 5.2), measure 0.84mL formic acid (concentration 88%), 3.43mL triethylamine, add to 1000mL pure water, use 2mol / L formic acid aqueous solution to adjust the pH to 5.2 to obtain formic acid-triethylamine aqueous solution; mix them in the ratio of methanol: triethylamine formate water = 13:2, and shake well.

[0132] 3. Chromatographic determination and chromatographic conditions:

[0133] The chromatographic conditions were the same as those in Example 1. The mixed standard solution prepared in this example was injected into the chromatograph for measurement, and its chromatogram was recorded. Figure 7 The separation degree is shown in Table 2. Figure 7 It can be seen that after pH adjustment, the overlap of LPPG, DPPA, and DPPC changed, and the retention time changed, but the separation degree of DPPG-Na was 2.71, which was still greater than 1.5, which means that within this pH range, the system is relatively stable.

[0134] Table 2 Separation of components in the mixed standard solution of Example 3

[0135] name DPPG-Na DPPA LPPG DPPC Separation 2.71 1.77 2.79 2.52

[0136] Comparative Example 1

[0137] The commonly used content detection methods for phospholipids at present are often referred to the phosphatidylcholine and phosphatidylethanolamine determination methods in the egg yolk lecithin (for injection) content determination item in the Chinese Pharmacopoeia. A mixed standard solution was prepared according to Example 1 and the mixed standard solution was tested. The chromatographic conditions were as follows: chromatographic column: Sepax HP-Silica 4.6×250mm 5μm; column temperature 40°C; flow rate: 1.0mL / min; injection volume 20μL; mobile phase A: methanol-water-glacial acetic acid-triethylamine (85:15:0.45:0.05), mobile phase B: n-hexane-isopropanol-mobile phase A=20:48:32, elution gradient: 0-20min, mobile phase A: 10%→30%, mobile phase B: 90%→70%; 20-35min, mobile phase A: 30%→95%, mobile phase B: 70%→5%; 35-36min, mobile phase A: 95%→10%, mobile phase B: 5%→90%; 36-41min, mobile phase A: 10%, mobile phase B: 90%. ELSD detector: drift tube 72℃, flow rate 2.0L / min, split flow, gain 1. The recorded spectrum is shown in the figure. Figure 8 shown.

[0138] There are 4 components in the mixed standard solution. Under the conditions of the phosphatidylcholine and phosphatidylethanolamine determination method in the egg yolk lecithin (for injection) content determination item, only 2 main peaks appear; and the peak area with a retention time of about 5 minutes is larger than the peak area with a retention time of about 19 minutes, which is considered to be formed by the stacking of DPPG-Na, DPPA, and LPPG-Na peaks. This indicates that in this chromatographic system, DPPG-Na is stacked with its impurities, and accurate content detection cannot be performed. A new detection method needs to be established to separate DPPG from its three impurity components before content determination.

[0139] Comparative Example 2

[0140] There are few methods for detecting DPPG-Na, and it can only be carried out by referring to other methods for detecting phospholipids. In addition to the method for detecting the content of egg yolk lecithin as a reference, the inventor has noticed a method for detecting 5 kinds of phospholipids in whey protein powder, which can separate PC, PE, PI, PS, and SM in the same system, meeting the requirement of separating DPPG-Na from other components in one system. Using the mixed standard solution of Example 1, detection was carried out under the following chromatographic conditions.

[0141] Chromatographic column: Sepax HP-Silica 4.6×250mm 5μm; column temperature 25℃; flow rate: 1.0mL / min; injection volume 20μL; accurately weigh 77.39mg of ammonium acetate solid and dissolve it in 1L of pure water to obtain 0.01mol / L ammonium acetate solution. Mobile phase A (95% acetonitrile ammonium acetate solution): measure 475mL acetonitrile, 25mL 0.01mol / L ammonium acetate solution, mix well, and adjust the pH to 5.6 with glacial acetic acid. Mobile phase B (50% acetonitrile ammonium acetate solution): measure 250mL acetonitrile, 250mL 0.01mol / L ammonium acetate solution, mix well, and adjust the pH to 5.6 with glacial acetic acid.

[0142] The elution gradient is: 0-3min, mobile phase A: 99%, mobile phase B: 1%; 3-7min, mobile phase A: 99%→91%, mobile phase B: 1%→9%; 7-10min, mobile phase A: 91%→88%, mobile phase B: 9%→12%; 10-20min, mobile phase A: 88%→75%, mobile phase B: 12%→25%; 20-25min, mobile phase A: 75%, mobile phase B: 25%; 25-27min, mobile phase A: 75%→99%, mobile phase B: 25%→1%; 27-35min, mobile phase A: 99%, mobile phase B: 1%. ELSD detector: drift tube 90℃, flow rate 2.2L / min, split flow, gain 1. Record the spectrum as follows Fig. 9 .

[0143] Although the method in Comparative Example 2 can separate DPPG-Na from LPPG-Na and DPPC, the peak areas of different components under this method vary greatly, and DPPG-Na is closer to the solvent peak, and the peak shape shows a tailing, indicating that the system is not conducive to DPPG-Na detection.

[0144] Comparative Example 3

[0145] With the increasing application of phospholipids as excipients, research on phospholipids has also increased. In addition to domestic research, foreign analysis and detection research on phospholipids is also gradually underway. In an article published in Journal of Chromatography B in 2020 (Normal phase HPLC method for combined separation of both polar and neutral lipid classes with application to lipid metabolic flux, J Chromatogr BAnalyt Technol Biomed Life Sci. 2020May 15:1145:122099.doi:10.1016 / j.jchromb.2020.122099.Epub 2020Apr 3.), there is a study on the use of HPLC system to separate various phospholipid components, claiming to be able to separate PG, PE, PI, PS, PC, LPC, etc. Use the mixed standard solution and blank solution of Example 1 to detect DPPG according to its conditions. The chromatographic conditions are as follows:

[0146] Chromatographic column: Sepax HP-Silica 4.6×250mm 5μm; column temperature 40℃; flow rate: 1.0mL / min; injection volume 20μL; accurately take 0.84mL formic acid and 2.5g triethylamine in 1L pure water, mix well, adjust pH to 4.6 to obtain methanol triethylamine aqueous solution as mobile phase D; mobile phase A: methanol; mobile phase B: n-hexane; mobile phase C: isopropanol. The elution gradient is: 0-3min, mobile phase A: 0.5%→1%, mobile phase B: 80%→25%, mobile phase C: 19.3%→73.6%, mobile phase D: 0.2%→0.4%; 3-6min, mobile phase A: 1%→1.5%, mobile phase B: 25%→10%, mobile phase C: 73.6%→87.5%, mobile phase D: 0.4%→1.0%; 6-11min, mobile phase A: 1.5 %→2%, mobile phase B: 10%→5%, mobile phase C: 87.5%→91.5%, mobile phase D: 1.0%→1.5%; 11-13 min, mobile phase A: 2%→12%, mobile phase B: 5%→0%, mobile phase C: 91.5%→83.2%, mobile phase D: 1.5%→4.8%; 13-15 min, mobile phase A: 12%→25%, mobile phase B: 0%, mobile phase C: 83.2% →65%, mobile phase D: 4.8%→10%; 15-18 min, mobile phase A: 25%, mobile phase B: 0%, mobile phase C: 65%, mobile phase D: 10%; 18-20 min, mobile phase A: 25%→0%, mobile phase B: 0%, mobile phase C: 65%→100%, mobile phase D: 10%→0%; 20-23 min, mobile phase A: 0%, mobile phase B: 0%, mobile phase C: 100%, Mobile phase D: 0%; 23-24 min, mobile phase A: 0% → 0.5%, mobile phase B: 0% → 80%, mobile phase C: 100% → 19.3%, mobile phase D: 0% → 0.2%; 24-28 min, mobile phase A: 0.5%, mobile phase B: 80%, mobile phase C: 19.3%, mobile phase D: 0.2%; ELSD detector: drift tube 72°C, flow rate 2.0 L / min, split flow, gain 1. Record the spectrum as follows Fig.10 and Fig.11 .

[0147] Fig.10 For mixed standard injection, it can be seen that the system cannot separate DPPC, and combined with Fig.11In the blank injection, an interference peak appeared near the retention time of the DPPG-Na peak. This system can separate a variety of phospholipids to a certain extent, but it will interfere with the system and may not be able to perform accurate quantitative detection. In addition, the mobile phase of this system occupies 4 liquid phase channels, and its mobile phase ratio varies greatly. The mobile phase ratio needs to be controlled in decimal places, which has high requirements for the instrument and causes great damage to the diverter valve. Considering the above situation, it is not suitable for the content detection of DPPG-Na.

[0148] Effect Example 1

[0149] Limit of detection and limit of quantitation:

[0150] In order to verify the detection limit and quantification limit of the detection method in the embodiment, take the DPPG-Na standard curve solution 1 in Example 1, accurately take 0.1mL in a 2mL volumetric flask, dilute to scale with chloroform: methanol: water (65: 35: 4), and obtain the quantification limit solution; accurately take 1mL of the quantification limit in a 2mL volumetric flask, dilute to scale with chloroform: methanol: water (65: 35: 4), and obtain the detection limit solution. Take the detection limit solution and inject 3 injections continuously, and the quantification limit solution and inject 6 injections continuously, and the data are shown in Tables 3 and 4.

[0151] Table 3 Detection limits

[0152]

[0153] Table 4 Limit of quantitation

[0154]

[0155] In this system, the detection limit of DPPG-Na was 2.635 μg / mL, the quantification limit was 5.27 μg / mL, and the RSD of the quantification limit after 6 consecutive injections was 3.46%, which was less than 5%.

[0156] Effect Example 2

[0157] Repeatability test:

[0158] In order to verify the repeatability of the detection method in the embodiment, DPPG-Na standard curve solutions 1 to 5 in Example 1 were taken as standard curves, and 6 test samples were prepared in parallel according to Example 1. The results are shown in Table 5. The content was 87.83%, and the RSD was 1.65%, which was less than 5%, indicating that the method was stable.

[0159] Table 5 Repeatability

[0160]

[0161] Effect Example 3

[0162] Recovery test:

[0163] In order to verify the accuracy of the present invention, a recovery test was conducted, and the DPPG-Na standard curve solutions 1 to 5 in Example 1 were taken as the standard curve. Preparation of recovery solution: Take the sample, accurately weigh 7.9 mg, 8.01 mg, and 7.91 mg in 50 mL volumetric flasks, numbered 1 to 3, add DPPG-Na control 7.76 mg, 7.70 mg, and 7.54 mg in 1 to 3 bottles, respectively, add chloroform: methanol: water (65:35:4) to dissolve and dilute to the scale, shake well, and use it as the test solution. The results are shown in Table 6. The recovery rate is between 100.2% and 104.77%. The above data show that the embodiment method can separate DPPG-Na and its impurities, and it has good repeatability and accurate recovery rate. It is a new method for determining the content of DPPG-Na.

[0164] Table 6 Recovery

[0165]

[0166] Tables 7 to 10 are information on the relevant test instruments, standards, reagents and samples used in the examples and comparative examples:

[0167] Table 7 Reagent information

[0168] Reagent name level factory Methanol HPLC Simga Isopropyl alcohol HPLC BCR n-Hexane HPLC BCR Triethylamine HPLC Maclean Formic acid AR Guangdong Guanghua Chloroform AR Hunan Huihong Methanol AR Guangdong Guanghua

[0169] Table 8 Instrument information

[0170] Instrument Name Manufacturer and Model Electronic balance Shimadzu AP225WD High performance liquid chromatography Thermo U3000 ELSD detector AllChrom ELSD 6100 Chromatographic columns Sepax HP-Silica 250*4.6mm 5μm

[0171] Table 9 Reference substance (standard substance) information

[0172] Reference source DPPG-Na Sigma DPPA Sigma LPPG-Na Baiyunshan Chinese Medicine DPPC Sigma

[0173] Table 10 Sample information used in the present invention

[0174] sample Preparation method DPPG-Na Baiyunshan Chinese Medicine

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting a salt of dipalmitoylphosphatidylglycerol, characterized in that: It includes separation of dipalmitoylphosphatidylglycerol salts in samples by high performance liquid chromatography; The HPLC detection conditions include: The chromatographic column adopts a silica gel chromatographic column; The mobile phase system includes mobile phase B, mobile phase C and mobile phase D; Mobile phase B was n-hexane, mobile phase C was isopropanol, and mobile phase D contained methanol and formic acid-triethylamine aqueous solution.

2. The detection method according to claim 1, characterized in that: The volume ratio of methanol to formic acid-triethylamine aqueous solution in mobile phase D is (12-14):2, preferably 13:2; Optionally, the pH of the formic acid-triethylamine aqueous solution is 4.8-5.

5.

3. The detection method according to claim 2, characterized in that: The formic acid-triethylamine aqueous solution was prepared as follows: 0.84 mL of formic acid and 3.43 mL of triethylamine were added to 1000 mL of pure water, and the pH was adjusted to between 4.8 and 5.2 using a formic acid aqueous solution to obtain a formic acid-triethylamine aqueous solution.

4. The detection method according to claim 1, characterized in that: Perform gradient elution. The gradient elution program is as follows: 0-1min, mobile phase B 80%→20%, mobile phase C 20%→72%, mobile phase D 0%→8%; 1-4 min, mobile phase B 20%, mobile phase C 72%, mobile phase D 8%; 4-8 min, mobile phase B 20%→25%, mobile phase C 72%→50%, mobile phase D 8%→25%; 8-12 min, mobile phase B 25%, mobile phase C 50%→45%, mobile phase D 25%→30%; 12-16 min, mobile phase B 25%, mobile phase C 45%→30%, mobile phase D 30%→45%; 16-23 min, mobile phase B 25% → 0%, mobile phase C 30% → 65%, mobile phase D 45% → 35%; 23-27 min, mobile phase B 0%, mobile phase C 65%, mobile phase D 35%; 27-28 min, mobile phase B 0%, mobile phase C 65%→100%, mobile phase D 35%→0%; 28-33 min, mobile phase B 0%, mobile phase C 100%, mobile phase D 0%; 33-34 min, mobile phase B 0%→80%, mobile phase C 100%→20%, mobile phase D 0%; 34-40 min, mobile phase B 80%, mobile phase C 20%, mobile phase D 0%.

5. The detection method according to claim 1, characterized in that: The column specifications are 250×4.6mm 5μm; Optionally, the chromatographic column uses a Sepax HP-Silica column with specifications of 250×4.6 mm 5 μm; Optionally, the column temperature of the chromatographic column is 35-45°C, preferably 40°C.

6. The detection method according to claim 1, characterized in that: The detector is an evaporative light scattering detector; Optionally, when the evaporative light scattering detector is used for detection, the drift tube temperature is 80° C. and the gas flow rate is 2.0 L / min.

7. The detection method according to claim 1, characterized in that: The solvents for the test solution were chloroform, methanol, and water; Optionally, the solvent of the test solution is chloroform, methanol and water in a volume ratio of 65:35:4; Optionally, the sample includes one or more of a sample to be tested, a standard product, and a quality control product; Optionally, the concentration of the sample to be tested in the test solution is 0.1-1 mg / mL, preferably 0.3 mg / mL.

8. The detection method according to claim 1, characterized in that: The detection method further comprises obtaining a chromatogram and calculating the content of the salt of dipalmitoylphosphatidylglycerol; Optionally, the content of the dipalmitoylphosphatidylglycerol salt is analyzed and calculated using an external standard method; Optionally, the external standard method is a standard curve method.

9. The detection method according to any one of claims 1 to 8, characterized in that: The dipalmitoylphosphatidylglycerol salt is dipalmitoylphosphatidylglycerol sodium; Optionally, the sample further contains one or more of dipalmitoylphosphatidic acid, a salt of lysopalmitoylphosphatidylglycerol, and dipalmitoylphosphatidylcholine; Optionally, the sample comprises synthetic phospholipids.

10. Use of the dipalmitoylphosphatidylglycerol salt detection method according to any one of claims 1 to 9 in any one of the following (i) to (iii): (i) preparing a salt of dipalmitoylphosphatidylglycerol or performing quality control on a salt of dipalmitoylphosphatidylglycerol; (ii) preparing phospholipids or performing quality control on phospholipids; (iii) Preparation of pharmaceutical excipients or quality control of pharmaceutical excipients.

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