阅读说明：本技术 一种高效液相色谱测定二肉豆蔻酰磷脂酰胆碱含量的方法 (Method for determining content of dimyristoyl phosphatidylcholine by high performance liquid chromatography ) 是由 王冬冬 宗玺 金晓婷 于文渊 刘来富 夏清 杨亮 胡海燕 吉民 于 2020-11-27 设计创作，主要内容包括：本发明公开了一种二肉豆蔻酰磷脂酰胆碱的高效液相色谱检测方法。其色谱条件为：色谱柱：用十八烷基键合硅胶为填充剂的色谱柱；流动相：A相为4mmol/L pH＝4的醋酸铵缓冲溶液,B相为4mmol/L醋酸铵甲醇溶液,按一定的梯度洗脱,使用蒸发光散射检测器(ELSD),进行高效液相色谱分析,测定二肉豆蔻酰磷脂酰胆碱的含量。本发明通过高效液相检测,运用反相色谱法,能够准确测出二肉豆蔻酰磷脂酰胆碱的含量,且与其他有关物质能够达到很好的分离效果,提高了检测准确性,且对检测仪器及色谱柱损害较小。(The invention discloses a high performance liquid chromatography detection method of dimyristoyl phosphatidylcholine. The chromatographic conditions are as follows: a chromatographic column: chromatographic column using octadecyl bonded silica gel as filler; mobile phase: the phase a was a 4mmol/L ammonium acetate buffer solution having a pH of 4, and the phase B was a 4mmol/L ammonium acetate methanol solution, and the content of dimyristoyl phosphatidylcholine was measured by high performance liquid chromatography using an Evaporative Light Scattering Detector (ELSD) with elution performed in a predetermined gradient. According to the invention, the content of dimyristoyl phosphatidylcholine can be accurately measured by high performance liquid detection and reverse phase chromatography, and the dimyristoyl phosphatidylcholine can be well separated from other related substances, so that the detection accuracy is improved, and the damage to a detection instrument and a chromatographic column is small.)

1. A method for measuring the content of dimyristoyl phosphatidylcholine by high performance liquid chromatography is characterized in that the chromatographic conditions are as follows: a chromatographic column: a high performance liquid reverse phase chromatography column; mobile phase: the phase A is ammonium acetate buffer solution, and the phase B is ammonium acetate methanol solution; evaporate the light scattering detector and elute in a gradient.

2. The method according to claim 1, characterized in that the mobile phase: the phase A is an ammonium acetate buffer solution with the pH value of 4-4.5 mmol/L, and the phase B is an ammonium acetate methanol solution with the pH value of 4 mmol/L.

3. The method according to claim 1, wherein the high performance liquid reverse phase chromatography column is a column using octadecyl bonded silica gel as a filler.

4. The method according to claim 2, characterized in that the gradient elution procedure is: and (3) changing the linear gradient of the mobile phase A and the mobile phase B from the initial volume ratio of 15:85 to 0:100 within 0-8 minutes, continuously eluting with 100% of the phase B to the 14 th minute, and continuously eluting with a mixed solution of the mobile phase A and the mobile phase B in the volume ratio of 15:85 to the end at the 14.01 minute.

5. The method of claim 1, wherein the evaporative light scattering detector has a detector temperature: 40-80 ℃ and the air flow rate is 1.5-2.0L/min.

6. The method according to claim 1, characterized in that the mobile phase flow rate: 1.0-1.3 mL/min.

7. The method of claim 1, wherein the column temperature of the chromatography column is: 35-45 ℃.

8. The method of claim 1, wherein dimyristoyl phosphatidylcholine is detected over a linear range of: 30-70 mu g/ml.

9. The method according to claim 1, wherein the injection volume is 5 to 15 μ L.

Technical Field

The invention relates to a method for determining dimyristoyl phosphatidylcholine content by using high performance liquid chromatography, belonging to the field of instrument analysis.

Background

Dimyristoyl phosphatidylcholine (DMPC) belongs to synthetic phospholipid, is widely applied to pharmaceutic adjuvants, is suitable for improving a preparation prescription, and is prepared into various liposome and phospholipid complexes. Dimyristoyl phosphatidylcholine has been applied to Visudyne (verteporfin liposome) and Abelect (amphotericin B phospholipid complex), has been recorded in the database of inactive pharmaceutical ingredients (adjuvants) of the U.S. food and drug administration, and has quality standards not loaded in the chinese pharmacopoeia, the U.S. pharmacopoeia and the european pharmacopoeia. At present, no dimyristoyl phosphatidylcholine content determination method exists in domestic and foreign documents.

Dimyristoylphosphatidylcholine is generally prepared by chemical synthesis. The commonly used industrial method uses myristic acid (myristic acid) and phosphorylcholine as starting materials, and the myristic acid and the phosphorylcholine are prepared through condensation reaction and purification. In the preparation process, myristic acid is used as a starting material and is also a degradation product of the product. In addition, lysophosphatidylcholine, which is a byproduct, is produced in the reaction, and is also a compound easily produced in the degradation process of the product. These two compounds interfere with the accuracy of the dimyristoyl phosphatidylcholine content assay. It is very necessary to develop a content detection method capable of separating dimyristoyl phosphatidylcholine and related substances thereof.

At present, TLC (thin layer chromatography), liquid chromatography detected by an ultraviolet detector, liquid normal phase chromatography detected by an ELSD (Electron cyclotron resonance) detector and the like are used for detecting the content of other phosphatidylcholine compounds at home and abroad. The TLC method cannot accurately and quantitatively detect the content of the product, and the content detection error is large due to large human subjectivity. The phospholipid compounds almost have no ultraviolet absorption, so the detection wavelength of the liquid chromatography detected by an ultraviolet detector is generally selected between 190 and 210nm, the interference of solvents and the like used by a mobile phase under the detection wavelength is large, the response value of a sample is low, and the repeatability and the accuracy of content detection are poor. In the quality standards of soybean lecithin and yolk lecithin in Chinese pharmacopoeia, an ELSD detector is used for detecting the content of phosphatidylcholine, a chromatographic column with silica gel as a filler is used for detecting the content of the phosphatidylcholine, normal phase chromatography with normal hexane, isopropanol, methanol and the like as solvents is used for detecting the content of the phosphatidylcholine, and the method has great damage to the chromatographic column and instruments. And the content detection of the phospholipid or the phosphatidylcholine compound does not specially aim at the content detection of dimyristoyl phosphatidylcholine.

Disclosure of Invention

Aiming at the situation that no method for measuring the content of dimyristoyl phosphatidylcholine exists at present, the invention provides a high performance liquid chromatography method for measuring dimyristoyl phosphatidylcholine, which can effectively solve the problems and accurately measure dimyristoyl phosphatidylcholine.

In order to achieve the purpose, the invention implements the following technical scheme:

a method for measuring the content of dimyristoyl phosphatidylcholine by high performance liquid chromatography comprises the following chromatographic conditions: a chromatographic column: a high performance liquid reverse phase chromatography column; mobile phase: the phase A is ammonium acetate buffer solution, and the phase B is ammonium acetate methanol solution; evaporate the light scattering detector and elute in a gradient.

Preferably, the chromatographic conditions are:

a chromatographic column: in the case of a column using octadecyl silica gel as a packing, it is preferable that the column has a length of 150mm, a diameter of 4.6mm, and a packing particle diameter of 3.5 μm, without affecting the detection effect. For example: agilent Eclipse Plus C18 column (4.6X 150mm, 3.5 μm).

Carrying out gradient elution by adopting a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the mobile phase is as follows: phase a was 4mmol/L ammonium acetate buffer at pH 4 and phase B was 4mmol/L ammonium acetate in methanol.

The gradient elution procedure was:

and (3) changing the linear gradient of the mobile phase A and the mobile phase B from the initial volume ratio of 15:85 to 0:100 within 0-8 minutes, continuously eluting with 100% of the phase B to the 14 th minute, and continuously eluting with a mixed solution of the mobile phase A and the mobile phase B in the volume ratio of 15:85 to the end at the 14.01 minute.

Specifically, the gradient elution conditions are shown in the following table:

preferred mobile phase flow rates: 1.0-1.3 mL/min;

preferred column temperature of the column: 35-45 ℃;

evaporative light scattering detector temperature: 40-80 ℃, gas flow rate: and (3) performing high performance liquid chromatography analysis at a rate of 1.5-2.0L/min to determine the content of dimyristoyl phosphatidylcholine.

The establishment process of the high performance liquid chromatography detection method of dimyristoyl phosphatidylcholine comprises the following steps:

step a, preparing linear standard solutions of dimyristoyl phosphatidylcholine with different concentrations;

b, measuring the linear standard solutions of the dimyristoyl phosphatidylcholine with different concentrations by using a high performance liquid chromatograph, recording the obtained peak areas, taking the natural logarithm of the peak areas of the standard solutions of the dimyristoyl phosphatidylcholine series as the ordinate, taking the natural logarithm of the corresponding concentrations of the standard solutions as the abscissa, making a standard curve, and performing linear regression to obtain a standard curve regression equation;

and c, preparing a dimyristoyl phosphatidylcholine test sample solution, measuring by using a high performance liquid chromatograph under the same measuring conditions of the standard solution, recording peak area, and substituting the peak area into the regression equation of the standard curve obtained in the step c for calculation to obtain the content of dimyristoyl phosphatidylcholine.

The concentrations of the dimyristoyl phosphatidylcholine linear standard solution are respectively as follows: 30. mu.g/ml, 40. mu.g/ml, 50. mu.g/ml, 60. mu.g/ml, 70. mu.g/ml, the amount of sample was 10. mu.l.

The invention has the advantages that:

selection of mobile phase: dimyristoyl phosphatidylcholine is dissolved in methanol and is almost insoluble in water, methanol is selected as a diluent of a sample solution, gradient elution is carried out by adopting a mixed mobile phase of a mobile phase A (4mmol/L ammonium acetate buffer solution with pH value of 4) and a mobile phase B (4mmol/L ammonium acetate methanol solution), a C18 column is adopted for separation, the polarity of the mobile phase is far greater than that of a stationary phase, and the method is reverse phase chromatography and has small damage to a chromatographic column and instruments; and the peak emergence time of each component is fast, and the peak emergence time of dimyristoyl phosphatidylcholine is about 11 min.

The high performance liquid chromatography determination method of dimyristoyl phosphatidylcholine provided by the invention has the characteristics of high accuracy and good stability. Under the detection condition of the invention, the peak time of dimyristoyl phosphatidylcholine is appropriate, main impurities of lysophosphatidylcholine and myristic acid are well separated from the product of myristoyl phosphatidylcholine, the content of the myristoyl phosphatidylcholine can be accurately determined, the impurities of lysophosphatidylcholine and myristic acid can be quantified, and the method has great significance for establishing the quality standard of dimyristoyl phosphatidylcholine and controlling impurities.

The analysis method disclosed by the invention has the advantages of good linearity within a certain concentration range, high precision, good repeatability and strong stability.

According to the chromatographic conditions selected by the invention, each component can be effectively separated and detected, a standard solution prepared from a dimyristoyl phosphatidylcholine standard substance is adopted for determination, a linear regression equation is calculated according to the method, the correlation coefficient r is 0.9994, the detection range is wide, the method disclosed by the invention is good in linearity within the concentration range of 30-70 mu g/ml, the precision is high, 6 times of parallel determination are carried out on the same sample, and the relative standard deviation of the peak area is only 0.22%.

Drawings

FIG. 1 is a chromatogram of the high performance liquid phase in example 1.

FIG. 2 is a line graph of a dimyristoylphosphatidylcholine standard solution of example 2.

FIG. 3 is a chromatogram of the high performance liquid phase of specific resolution in example 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following method screening is performed in combination with specific examples, and the present invention is further described in detail. It should be understood that the specific examples described herein are intended to be illustrative only and are not intended to be limiting. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Example 1: high performance liquid chromatography determination method I of dimyristoyl phosphatidylcholine

Experimental materials and instruments

Control, sample and reagent: dimyristoyl phosphatidylcholine (DMPC) reference (with a Lipoid content of 99.6%), lysophosphatidylcholine (jiangsu southeast nanomaterial co., ltd.), myristic acid (lingfeng chemical), chromatographic Tetrahydrofuran (TEDIA), and trifluoroacetic acid (national drug group).

The instrument comprises the following steps: the name and model of the specific instrument are shown in table 1 below.

TABLE 1 name and model of the specific instrument

Name of instrument	Model number
		Analytical balance	SQP/SECURA225D-1CN
High performance liquid chromatograph	Agilent1260
		ELSD detector	Ontai 6000
Acidimeter	PHS-3C

Liquid chromatography conditions:

the chromatographic column adopts Kromasil 100-5-C18 column (4.6 x 150 mm); and (2) adding tetrahydrofuran: water: isocratic elution is carried out for 30 minutes by taking trifluoroacetic acid as a mobile phase with a ratio of 900:10: 1; the column temperature is 40 ℃; the evaporative light scattering detector temperature was 45 ℃ and the nitrogen flow rate was 1.7L per minute.

The experimental process comprises the following steps:

taking appropriate amount of dimyristoyl phosphatidylcholine control, lysophosphatidylcholine and myristic acid, dissolving with methanol, and diluting to obtain mixed solution containing the control 0.05mg, 0.01mg and 0.01mg in each 1 mL. Precisely measuring 10 μ L of the mixed solution, injecting into a liquid chromatograph, and recording chromatogram.

The test results are shown in FIG. 1. The results showed that the separation of myristic acid from lysophosphatidylcholine was 0.42, and that the separation of lysophosphatidylcholine from dipalmitoyl phosphatidylcholine was 0.52, both of which were not effective.

Example 2: high performance liquid chromatography method II for measuring dimyristoyl phosphatidylcholine (measuring method of the present invention)

Experimental materials and instruments

Control, sample and reagent: dimyristoyl phosphatidylcholine control (Lipoid content 99.6%), lysophosphatidylcholine (Jiangsu southeast nanomaterial Co., Ltd.), myristic acid (Lingfeng Chemicals), dimyristoyl phosphatidylcholine (Jiangsu southeast nanomaterial Co., Ltd.), chromatographic methanol (TEDIA), and ammonium acetate (Lingfeng Chemicals).

The test apparatus was the same as in example 1.

Liquid chromatography conditions:

the column was an Agilent Eclipse Plus C18 column (4.6 x 150mm, 3.5 μm); taking 4mmol/L ammonium acetate buffer solution with pH value of 4 as a mobile phase A, and taking 4mmol/L ammonium acetate methanol as a mobile phase B; gradient elution was performed as in table 2 below; the column temperature is 40 ℃; the light scattering detector was evaporated at 70 ℃ and nitrogen flow rate was 1.8L per minute.

The experimental process comprises the following steps:

taking about 5mg of the product, accurately weighing, placing in a 100mL measuring flask, adding methanol to dissolve and dilute to a scale, and shaking up to obtain a test solution. An appropriate amount of dimyristoyl phosphatidylcholine control was precisely weighed and diluted with methanol to prepare a solution containing dimyristoyl phosphatidylcholine 0.03mg, 0.04mg, 0.05mg, 0.06mg, and 0.07mg per 1mL as a standard solution. Precisely measuring 10 μ L of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording chromatogram. The dimyristoyl phosphatidylcholine content was calculated from the linear regression equation measured for the standard solution.

The standard solution linear regression equation is shown in figure 2. The three batches of sample detection data are shown in Table 2

TABLE 2

The result shows that the detection method disclosed by the invention has good linear and parallel detection results of each batch of samples, the content measurement result meets the requirement, and the method is suitable for detecting the content of dimyristoyl phosphatidylcholine products.

Example 3

In order to verify the feasibility and accuracy of the above example 2, a specificity test, a precision test, a sample solution stability test, and a linear relationship test were further performed. The materials and apparatus used in the test were the same as those in example 2.

1. Specificity

Solution preparation:

blank solution: methanol.

A special solution: taking appropriate amount of dimyristoyl phosphatidylcholine, lysophosphatidylcholine and myristic acid, respectively, dissolving with methanol, and diluting to obtain mixed solution containing the above control substances 0.05mg, 0.01mg and 0.01mg in each 1 mL.

Lysophosphatidylcholine localization solution: precisely weighing a lysophosphatidylcholine reference substance in a volumetric flask of 1mg to 100mL, adding a proper amount of methanol to dissolve, diluting with a diluent to a constant volume to scale, and uniformly mixing.

Myristic acid positioning solution: accurately weighing 1mg of the myristic acid to a 100mL volumetric flask, adding a proper amount of methanol to dissolve, diluting with a diluent to a constant volume to a scale, and uniformly mixing.

Dimyristoyl phosphatidylcholine localization solution: precisely weighing dimyristoyl phosphatidylcholine 5mg to 100mL in a volumetric flask, adding a proper amount of methanol to dissolve, then metering to the scale with methanol, and uniformly mixing.

After the system was equilibrated, 10 μ L of each solution was injected into a liquid chromatograph, and the chromatogram was recorded, as shown in fig. 3. The results are recorded in table 3.

TABLE 3 results of the specificity test

The test results show that: under the chromatographic condition, a solvent methanol is selected as a detection solvent, the baseline is stable, the solvent does not interfere the detection of the product, the number of the theoretical plates of the dimyristoyl phosphatidylcholine peak is 66452, the separation degree of dimyristoyl phosphatidylcholine and adjacent peaks in a mixed solution is good (both are above 1.5, see figure 3), and the specificity is good. The retention time of dimyristoyl phosphatidylcholine is about 11min, and the peak appearance time is good.

2. Precision-repeatability

Solution preparation: precisely weighing dimyristoyl phosphatidylcholine 5mg to 100mL in a volumetric flask, adding a proper amount of methanol to dissolve, then metering to the scale with methanol, and uniformly mixing. And respectively injecting 10 mu L of the mixture into a liquid chromatograph, introducing the mixture for 6 times, recording all peak areas, and calculating the RSD (%) of the natural logarithm of all the peak areas. The results are shown in Table 4.

TABLE 4 repeatability test data

Time	Repeatability-1	Repeatability-2	Repeatability-3	Repeatability-4	Repeatability-5	Repeatability-6	RSD(％)
								Peak area	1008.85	990.3	991.06	991.99	999.69	1028.02	/
Area of lg peak	3.0038	2.9958	2.9961	2.9965	2.9999	3.0120	0.22

The results show that: the sample repeatability test has the advantages that the sample injection peak area is about 1000, the lg peak area is about 3.0, the lg peak area RSD is 0.22%, and the method has good precision.

3. Sample solution stability test

Precisely weighing a sample to be tested in a volumetric flask of 5mg to 100mL, adding a proper amount of methanol to dissolve the sample, then fixing the volume to a scale with the methanol, uniformly mixing to prepare a sample solution of 0.05mg/mL, respectively injecting samples 0h, 2h, 4h, 6h and 8h after preparation, recording peak areas, and calculating RSD (%) of natural logarithm of peak areas at all time points. The results are shown in Table 5.

TABLE 5 sample solution stability test

The results show that: the peak area of the sample solution is about 1000 in 8 hours of standing, the area of lg peak is about 3.0, the RSD of lg peak is 0.17%, and the sample solution has good stability in 8 hours.

4. Linearity

Preparing dimyristoyl phosphatidylcholine standard stock solution: precisely weighing a dimyristoyl phosphatidylcholine reference substance in a volumetric flask of 5mg to 10mL, adding a proper amount of methanol to dissolve, diluting with methanol to a constant volume to a scale, and uniformly mixing. As a standard stock solution.

Linearity: respectively and precisely transferring 0.6mL, 0.8mL, 1.0mL, 1.2mL and 1.4mL of dimyristoyl phosphatidylcholine standard stock solution into a 10mL measuring flask, fixing the volume with methanol, and shaking up to obtain a series of standard solutions with concentration. Precisely sucking 10 μ l of each solution with gradient concentration from low concentration to high concentration, sequentially sampling and analyzing, recording chromatogram, performing linear regression with the natural logarithm of the concentration of dimyristoyl phosphatidylcholine as abscissa and the natural logarithm of the peak area of the reference as ordinate, and calculating regression equation, with the result shown in Table 6 and FIG. 2.

TABLE 6 results of linear investigation

The result shows that the sample concentration is in the range of 30-70 mu g/ml, the correlation coefficient R is 0.9994, and the linearity is good.

The results of the above embodiments show that the detection method of the invention has the advantages of good specificity, good linear relation, good solution stability, good precision and accurate and reliable detection result.