Method for determining content of soybean phosphatidylcholine or phosphatidyl ethanolamine in soft capsule

Abstract

The invention belongs to the field of substance content detection, and discloses a method for determining the content of soybean phosphatidylcholine or phosphatidylethanolamine in a soft capsule, which comprises the following steps: (1) preparing a soybean phosphatidylcholine or phosphatidylethanolamine standard solution; (2) preparing a to-be-detected sample solution; (3) performing high performance liquid chromatography analysis; (4) drawing a standard curve; and (5) calculating the content of soybean phosphatidylcholine or phosphatidyl ethanolamine. The method has the advantages of high response value, short retention time and stable base line, and is not interfered by other components in the soft capsule; the method has the advantages of simple operation, high precision and good repeatability, the recovery rate meets the analysis requirement, and the method can efficiently, quickly and accurately detect the content of two components, namely soybean phosphatidylcholine and phosphatidyl ethanolamine, of soybean phospholipid in the soft capsule content.

Description

Technical Field

[0001] The invention belongs to the field of substance content detection, and specifically relates to a method for determining the contents of soybean phosphatidylcholine or phosphatidylethanolamine, two components of soybean lecithin in soft capsules by high performance liquid chromatography. Background Art

[0002] Phospholipids are important biochemical intermediates that maintain the normal growth and life activities of plant and animal cells. Phospholipids are contained in the cells of almost all organisms. Plant lecithin mainly comes from oil seeds such as soybeans, rapeseeds, sunflower seeds, etc. Its types mainly include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), etc. Phospholipids are a class of phosphorus-containing lipid physiologically active substances. They have special physiological functions such as promoting cell growth, promoting fat metabolism, regulating blood lipids, strengthening brain and intelligence, immune regulation and anti-tumor. They are important components of human and many animal and plant tissues and play an important role in life activities. Phospholipids are natural amphoteric surfactants with emulsification, dispersion, wetting, demoulding, separation and other functions. Refined natural lecithin can be used as an excipient for the preparation of various pharmaceutical preparations. Soybean lecithin is used as an emulsifier in emulsion drugs. Emulsion drugs have large dispersion, fast absorption, fast onset, and targeting, which is conducive to improving bioavailability. Since lecithin is non-toxic, non-irritating, has good biocompatibility, has good physiological activity, and is a natural excellent emulsifier, lecithin is the first choice as an emulsifier when preparing emulsion drugs. Lecithin with high PC content is often used to prepare oil-in-water (O / W) emulsions, such as fat emulsions, anticancer drug Brucea javanica oil emulsions, etc. Soybean lecithin is used as a liposome membrane material to prepare liposomes. Liposomes have the characteristics of targeting and lymphatic orientation, sustained release, reduced drug toxicity, and improved stability of the main drug. They are often used as carriers of anti-tumor drugs, antiparasitic drugs, antibacterial drugs, hormone drugs, etc. in clinical practice. Lecithin is an important material for forming liposomes. Soybean lecithin is used as a stabilizer for suspensions, gas dispersants, solid dispersions, etc. Lecithin is a natural amphoteric surfactant with emulsification, dispersion, wetting, demoulding, separation, etc. Therefore, it is also often used in suspensions, gas dispersants, solid dispersions, etc., to play the role of suspending, dispersing, lubricating, etc. Soybean lecithin plays an important role in the preparation and efficacy of soft capsule contents. Soybean phosphatidylcholine or phosphatidylethanolamine is the most important component of soybean lecithin. Therefore, the analysis of the content of soybean phosphatidylcholine or phosphatidylethanolamine in soft capsule contents is particularly important.

[0003] At present, there are many methods for analyzing the content of phospholipids, mainly including acetone insoluble matter method, phosphorus measurement method (including molybdenum blue colorimetry, wet oxidation method), ultraviolet visible spectrophotometry, thin layer chromatography, high performance liquid chromatography (HPLC), mass spectrometry, etc. Among them, the values ​​measured by the acetone insoluble matter method also include non-phosphorus glycolipids, etc., which can only roughly reflect the content of phospholipids; phosphorus measurement method and ultraviolet visible spectrophotometry cannot accurately determine the relative content of different types of phospholipids (such as soybean phosphatidylcholine, phosphatidylethanolamine, etc.); thin layer chromatography detects lower content of the test substance, and the specificity and precision are not good. When using HPLC-MS / MS for quantification, the analyte must be ionized first, and compounds that are difficult to ionize cannot be detected. The requirements for instruments and reagents are very high, and the corresponding cost is very high.

[0004] National standards GB5009.272-2016, GB / 35867-2018, and the Chinese Pharmacopoeia all use high performance liquid chromatography to determine the content of different types of phospholipids, and all use n-hexane, isopropanol, and weak acid as mobile phases. More importantly, phosphatidylethanolamine peaks very early under these mobile phases and will be interfered by other components in the soft capsule contents, while soybean phosphatidylcholine peaks for a long time.

[0005] In view of the above reasons, it is necessary to further improve the chromatographic analysis conditions in the national standard and develop a method to optimize the content of soybean phosphatidylcholine and phosphatidylethanolamine, two components of soybean lecithin in the contents of soft capsules. Summary of the invention The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a method for determining the contents of soybean phosphatidylcholine and phosphatidylethanolamine, two components of soybean lecithin, in the contents of soft capsules by high performance liquid chromatography, which has higher specificity, precision and accuracy and better reproducibility.

[0006] The purpose of the present invention can be achieved through the following technical solutions: A method for determining the content of soybean phosphatidylcholine or phosphatidylethanolamine in a soft capsule comprises the following steps: (1) Weigh soybean phosphatidylcholine standard and phosphatidylethanolamine standard respectively, dissolve them in solvent and make up to volume, and prepare soybean phosphatidylcholine standard solution and phosphatidylethanolamine standard solution with different concentration gradients respectively; (2) Weigh the sample to be tested from the contents of the soft capsule, dissolve it with a solvent and make it constant, filter it and take the filtrate to obtain a sample solution to be tested; (3) The standard solution and the sample solution to be tested are sampled and analyzed by a high performance liquid chromatograph-ultraviolet detector, the soybean phosphatidylcholine or phosphatidylethanolamine component is qualitatively analyzed according to the retention time of the standard, and the peak area of ​​the soybean phosphatidylcholine or phosphatidylethanolamine is determined; wherein: The chromatographic conditions for the analysis of soybean phosphatidylcholine by high performance liquid chromatography include: using a silica gel column, using methanol-water-glacial acetic acid-triethylamine as mobile phase A and n-hexane-isopropanol-mobile phase A as mobile phase B for isocratic elution, and then detecting with an ultraviolet detector; The chromatographic conditions for the sample injection analysis of phosphatidylethanolamine by high performance liquid chromatography include: using an amino-bonded silica gel column, using a methanol solution-acetonitrile as a mobile phase for isocratic elution, and then detecting with an ultraviolet detector; (4) Determine the standard solution with different concentration gradients obtained in step (1) according to the method of step (3), with the concentration of soybean phosphatidylcholine or phosphatidylethanolamine in the standard solution as the horizontal axis and the peak area of ​​the measured standard soybean phosphatidylcholine or phosphatidylethanolamine as the vertical axis, and obtain the standard curve and standard curve equation by linear regression; (5) Determine the sample solution obtained in step (2) according to the method of step (3), substitute the peak area of ​​the soybean phosphatidylcholine or phosphatidylethanolamine of the sample to be tested obtained into the standard curve equation, calculate the concentration of soybean phosphatidylcholine or phosphatidylethanolamine in the sample solution to be tested, and then calculate the mass fraction of soybean phosphatidylcholine or phosphatidylethanolamine in the sample to be tested, thereby achieving the determination of the soybean phosphatidylcholine or phosphatidylethanolamine content in the soft capsule.

[0007] In the above determination method, further, in steps (1) and (2), the solvent is a chloroform-methanol solution, wherein the volume ratio of chloroform to methanol is 1 to 4:1. When the solubility of soybean phosphatidylcholine or phosphatidylethanolamine in chloroform is significantly greater than that in the mobile phase, the front peak may appear. Using chloroform-methanol solution as a solvent reduces the difference in solubility of soybean phosphatidylcholine or phosphatidylethanolamine in the solvent and the mobile phase, and improves the peak shape of the chromatographic peak.

[0008] Furthermore, in step (1), the preparation method of preparing soybean phosphatidylcholine standard solution and phosphatidylethanolamine standard solution with different concentration gradients comprises the following steps: weighing soybean phosphatidylcholine standard and phosphatidylethanolamine standard respectively, dissolving them with chloroform-methanol solution to prepare standard stock solution, and then quantitatively diluting with the chloroform-methanol solution to prepare standard solution with different concentration gradients, wherein the concentration gradient of soybean phosphatidylcholine or phosphatidylethanolamine in the standard solution is 10 μg / mL~200 μg / mL (for example, each 1 mL contains 10 μg, 20 μg, 40 μg, 100 μg, 200 μg of soybean phosphatidylcholine or phosphatidylethanolamine, respectively).

[0009] Furthermore, in step (2), the filter head used for filtration is a 0.45 μm nylon 66 filter head, which is used to remove fine particles and avoid damage to the liquid phase instrument and chromatographic column.

[0010] Furthermore, in step (3), the chromatographic conditions for the analysis of soybean phosphatidylcholine by high performance liquid chromatography also include: the size of the silica gel column is 4.6 mm × 250 mm, the column temperature is 35-45°C, the injection volume is 10-50 μL; the wavelength of the ultraviolet detector is 205 nm. The silanol group of the silica gel column has a polar interaction with the carbonyl group of phosphatidylcholine, and the ethylamine in the mobile phase A reduces the tailing of the phosphatidylcholine peak.

[0011] Furthermore, in step (3), in the mobile phase A, the volume ratio of methanol, water, glacial acetic acid and triethylamine is 80-90:10-20:0.45:0.05; in the mobile phase B, the volume ratio of n-hexane, isopropanol and mobile phase A is 17-23:45-51:32; the volume ratio of mobile phase A to mobile phase B is (30-50):(70-50), and the flow rate is 0.5-2 mL / min.

[0012] Furthermore, in step (3), the chromatographic conditions for the injection analysis of phosphatidylethanolamine by high performance liquid chromatography also include: the specifications of the amino-bonded silica gel column are 4.6 mm×250 mm, the column temperature is 35-45° C., and the injection volume is 20-100 μL. The amino group of the amino column has a polar interaction with the phosphate group of phosphatidylethanolamine, and the triethylamine in the mobile phase reduces the tailing of the phosphatidylethanolamine peak.

[0013] Furthermore, in step (3), the methanol solution is obtained by mixing glacial acetic acid, triethylamine and methanol in a volume ratio of 0.40-0.50:0.05:100; in the methanol solution-acetonitrile, the volume ratio of methanol solution to acetonitrile is (15-35): (85-65), and the flow rate is 0.5-2 mL / min.

[0014] Furthermore, in step (5), with the soybean phosphatidylcholine concentration X1 in the standard solution as the horizontal coordinate and the peak area of ​​phosphatidylcholine Y1 as the vertical coordinate, the linear regression obtained the standard curve equation: Y1= 4872.287 X1-4919.591, and the correlation coefficient r1 was 0.999; with the phosphatidylethanolamine concentration X2 in the standard solution as the horizontal coordinate and the peak area of ​​phosphatidylethanolamine Y2 as the vertical coordinate, the linear regression obtained the standard curve equation: Y2= 35111.400 X2+75330.667, and the correlation coefficient r2 was 0.999.

[0015] Furthermore, in step (5), the mass fraction of soybean phosphatidylcholine or phosphatidylethanolamine in the sample to be tested is calculated as M= ; Wherein, M is the content of soybean phosphatidylcholine or phosphatidylethanolamine in the sample to be tested, unit: %; c is the concentration of soybean phosphatidylcholine or phosphatidylethanolamine in the sample solution to be tested calculated in step (5), unit: μg / mL; V is the total volume of the sample solution to be tested prepared in step (2), unit: mL; m is the mass of the sample to be tested weighed in step (2), unit: mg.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention can efficiently determine the contents of two components of soybean lecithin (soy phosphatidylcholine and phosphatidylethanolamine) in the contents of soft capsules, with high specificity, precision and accuracy, and good reproducibility.

[0017] 2. The present invention optimizes the chromatographic analysis conditions by examining different mobile phase systems; a silica gel column and an isocratic elution system in which the mobile phase A is methanol-water-glacial acetic acid-triethylamine (85:15:0.45:0.05) and the mobile phase B is n-hexane-isopropanol-mobile phase A (20:48:32) are used to detect soybean phosphatidylcholine. Under this mobile phase, the response value of soybean phosphatidylcholine in the sample is high, the retention time is short, the baseline is stable, and it is not interfered by other components in the soft capsule; an amino-bonded silica gel column and a methanol solution (take Phosphatidylethanolamine was detected by an isocratic elution system of acetonitrile (a mixture of 0.45 mL of glacial acetic acid, 0.05 mL of triethylamine and 100 mL of methanol). Under this mobile phase, the retention time of phosphatidylethanolamine in the sample was short, the baseline was stable, and it was not interfered by other components in the soft capsule. Then, an ultraviolet detector was used in combination with an external standard peak area method for quantification. This method has high precision, good repeatability, and the recovery rate meets the analysis requirements. It can efficiently, quickly and accurately detect the contents of soybean phosphatidylcholine and phosphatidylethanolamine, two components of soybean lecithin, in the contents of soft capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the chromatogram of soybean phosphatidyl phospholipids in Example 1 of the present invention.

[0020] Figure 2 It is the linear regression equation of the standard solution in Example 1 of the present invention.

[0021] Figure 3 This is the chromatogram of phosphatidylethanolamine in Example 2 of the present invention.

[0022] Figure 4 It is the linear regression equation of the standard solution in Example 2 of the present invention.

[0023] Figure 5 It is the chromatogram of soybean lecithin in Comparative Example 1 of the present invention.

[0024] Figure 6 It is a chromatogram of the contents of the progesterone soft capsule in Comparative Example 1 of the present invention.

[0025] Figure 7 It is an overlay diagram of the blank solvent, positioning solution, mixed solution and soft capsule contents in Comparative Example 2 of the present invention.

[0026] Figure 8 It is an overlay diagram of the blank solvent, positioning solution and mixed solution in Comparative Example 3 of the present invention.

[0027] Fig. 9 It is an overlay diagram of the blank solvent, positioning solution and mixed solution in Comparative Example 4 of the present invention.

[0028] Fig.10 This is an overlay of the phosphatidylethanolamine localization solution in Comparative Example 5 of the present invention in different mobile phases. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0030] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0032] Experimental materials and instruments: Standard substances, test samples and reagents: contents of soft capsules (Hunan Chunkang Pharmaceutical Technology Co., Ltd., test samples), soybean phosphatidylcholine (PC) standard (China Food and Drug Inspection Institutes, content 97.3%), phosphatidylethanolamine (soybean) standard (PE) (China Food and Drug Inspection Institutes, content 99.6%), chloroform (Tianjin Fuyu, AR), methanol (Starmark, HPLC), acetonitrile (Starmark, HPLC), n-hexane (B&K, HPLC), isopropanol (Chengdu Kelong, HPLC), glacial acetic acid (Tianjin Fuyu, AR), triethylamine (Tianjin Fuyu, AR), and ultrapure water (homemade).

[0033] Instruments: The names and models of specific instruments are shown in Table 1.

[0034] Table 1: Names and models of instruments used

[0035] Embodiment 1: A method for determining the content of soybean phosphatidylcholine in soybean lecithin in soft capsules by high performance liquid chromatography, the operating steps are as follows: Step 1. Preparation of soybean phosphatidylcholine standard stock solution: accurately weigh 10 mg of soybean phosphatidylcholine, dissolve it with chloroform-methanol solution (volume ratio 2:1), transfer it to a 50 mL volumetric flask, and dilute it to the mark with chloroform-methanol solution (volume ratio 2:1) to prepare a soybean phosphatidylcholine standard stock solution with a concentration of 200 μg / mL (also used as a standard solution); Step 2, preparation of soybean phosphatidylcholine standard solution: take 1mL of standard stock solution and place it in 20mL, 10mL and 5mL volumetric flasks respectively, take 5mL of standard stock solution and place it in 10mL volumetric flask, add chloroform-methanol solution (volume ratio 2:1) to make up to the scale, prepare a series of soybean phosphatidylcholine standard solutions with a concentration gradient of 10μg / mL~100μg / mL, and obtain standard solutions containing 10μg, 20μg, 40μg, 100μg and 200μg of soybean phosphatidylcholine per 1mL; Step 3, preparation of the sample solution to be tested: accurately weigh 2000 mg of the sample to be tested from the soft capsule content, place it in a 10 mL volumetric flask, add chloroform-methanol solution (volume ratio 2:1) to dissolve and dilute to the scale, prepare a test solution with a soybean phosphatidylcholine content within the concentration range of the series of standard solutions, filter with a 0.45 μm nylon 66 filter head, and take the filtrate; Step 4, high performance liquid chromatography analysis: the standard solution and the sample solution to be tested are analyzed by high performance liquid chromatography-ultraviolet detector, the soybean phosphatidylcholine component is qualitatively analyzed according to the retention time of the standard (the main peak retention time is about 8 minutes), and the peak area of ​​soybean phosphatidylcholine is determined; Soybean phosphatidylcholine liquid chromatography conditions: RD-SiO2 silica gel column (4.6×250mm, 5μm), column temperature 40℃, injection volume 10μL, mobile phase A methanol-water-glacial acetic acid-triethylamine (volume ratio 85:15:0.45:0.05), mobile phase B n-hexane-isopropanol-mobile phase A (volume ratio 20:48:32), elution with mobile phase A-mobile phase B (volume ratio 40:60), flow rate 1.0mL / min, UV detector detection wavelength 205nm; the high performance liquid chromatogram of the sample to be tested is shown as follows Figure 1 As shown (the interference peaks of other components of the soft capsule are before 6 minutes), it shows that the response value of soybean phosphatidylcholine in the sample is high, the retention time is short, the baseline is stable, and it is not interfered by other components in the soft capsule; Step 5, drawing of standard curve: for a series of standard solutions measured under the above chromatographic conditions, with the soybean phosphatidylcholine concentration X1 in the standard solution as the abscissa and the peak area of ​​phosphatidylcholine Y1 as the ordinate, linear regression is performed to obtain the standard curve (such as Figure 2 As shown) and the linear regression equation: Y1= 4872.287 X1-4919.591, the correlation coefficient r1 is 0.999; Step 6, calculation of soybean phosphatidylcholine content: Substitute the peak area of ​​phosphatidylcholine in the sample to be tested (about 240,000, which meets the requirement of accurate content calculation) into the linear regression equation of the standard curve to calculate the concentration of phosphatidylcholine in the sample to be tested, and then use the formula M= Calculate the mass fraction of phosphatidylcholine in the sample to be tested, where M is the content of soybean phosphatidylcholine or phosphatidylethanolamine in the sample, in units of %; c is the concentration of soybean phosphatidylcholine or phosphatidylethanolamine in the sample solution to be tested calculated according to step 5, in units of μg / mL; V is the total volume of the sample solution to be tested prepared in step 3, in units of mL; and m is the mass of the sample to be tested weighed in step 3, in units of mg.

[0036] In this embodiment, the mass fraction of phosphatidylcholine in the sample to be tested is calculated to be 0.029%.

[0037] Embodiment 2: A method for determining the phosphatidylethanolamine content of soybean lecithin in soft capsules by high performance liquid chromatography, the operating steps are as follows: Step 1. Preparation of phosphatidylethanolamine standard stock solution: accurately weigh 10 mg of phosphatidylethanolamine, dissolve it with chloroform-methanol solution (volume ratio 2:1), transfer it to a 50 mL volumetric flask, and dilute it to the mark with chloroform-methanol solution (volume ratio 2:1) to prepare a phosphatidylethanolamine standard stock solution with a concentration of 200 μg / mL (also used as a standard solution); Step 2, preparation of phosphatidylethanolamine standard solution: take 1mL of standard stock solution and place it in 20mL, 10mL and 5mL volumetric flasks respectively, take 5mL of standard stock solution and place it in 10mL volumetric flask, add chloroform-methanol solution (volume ratio 2:1) to make up to the scale, prepare a series of phosphatidylethanolamine standard solutions with a concentration gradient of 10μg / mL to 100μg / mL, and obtain standard solutions containing 10μg, 20μg, 40μg, 100μg and 200μg of soybean phosphatidylcholine per 1mL; Step 3, preparation of the sample solution to be tested: accurately weigh 2000 mg of the sample to be tested from the soft capsule content, place it in a 10 mL volumetric flask, add chloroform-methanol solution (volume ratio 2:1) to dissolve and dilute to the scale, prepare a solution to be tested with a phosphatidylethanolamine content within the concentration range of the series of standard solutions, filter with a 0.45 μm nylon 66 filter, and take the filtrate; Step 4, high performance liquid chromatography analysis: the standard solution and the sample solution to be tested are analyzed by high performance liquid chromatography-ultraviolet detector, the phosphatidylethanolamine component is qualitatively analyzed according to the retention time of the standard (the main peak retention time is about 17 minutes), and the peak area of ​​phosphatidylethanolamine is determined; Phosphatidylethanolamine liquid chromatography conditions: Sepax Hp-Amino amino-bonded silica gel column (4.6×250mm, 5μm), column temperature 40°C, injection volume 50μL, elution with methanol solution (mixed with 0.45mL glacial acetic acid, 0.05mL triethylamine and 100mL methanol)-acetonitrile (25:75), flow rate 1.0mL / min, UV detector detection wavelength 205nm; the high performance liquid chromatogram of the sample to be tested is as follows Figure 3 As shown (the interference peaks of other components of the soft capsule are before 12 minutes), the retention time of phosphatidylethanolamine in the sample is short, the baseline is stable, and it is not interfered by other components in the soft capsule; Step 5, drawing of standard curve: for a series of standard solutions measured under the above chromatographic conditions, with the concentration of phosphatidylethanolamine in the standard solution X2 as the abscissa and the peak area of ​​phosphatidylethanolamine Y2 as the ordinate, linear regression is performed to obtain the standard curve (such as Figure 4 As shown) and the linear regression equation: Y2= 35111.400 X2+75330.667, the correlation coefficient r2 is 0.999; Step 6, calculation of phosphatidylethanolamine content: Substitute the peak area of ​​phosphatidylethanolamine in the sample to be tested (about 750,000) into the linear regression equation of the standard curve to calculate the concentration of phosphatidylethanolamine in the sample to be tested, and then use the formula M= Calculate the mass fraction of phosphatidylethanolamine in the sample, where M is the content of phosphatidylethanolamine or phosphatidylethanolamine in the sample, in units of %; c is the concentration of phosphatidylethanolamine or phosphatidylethanolamine in the sample solution to be tested calculated according to step 5, in units of μg / mL; V is the total volume of the sample solution to be tested prepared in step 3, in units of mL; m is the mass of the sample to be tested weighed in step 3, in units of mg; In this embodiment, the mass fraction of phosphatidylethanolamine in the sample is calculated to be 0.010%.

[0038] Embodiment 3: Precision investigation: A soybean phosphatidylcholine (abbreviated as PC) standard solution with a concentration of 200 μg / mL was injected 5 times under the chromatographic conditions of Example 1; a phosphatidylethanolamine (abbreviated as PE) standard solution with a concentration of 200 μg / mL was injected 5 times under the chromatographic conditions of Example 2. The peak areas are shown in Table 2 below: Table 2: Peak area results of precision investigation experiment

[0039] After calculation, the relative standard deviation (RSD, the same below) of soybean phosphatidylcholine was 3.2%, and the RSD of phosphatidylethanolamine was 0.9%, indicating that the method has good precision.

[0040] Embodiment 4: Repeatability investigation: Take the sample solution to be tested and inject it 5 times under the chromatographic conditions of Example 1 and Example 2 respectively; the peak areas are shown in Table 3 below: Table 3: Peak area results of repeatability test

[0041] The calculated RSD of soybean phosphatidylcholine was 4.8%, and that of phosphatidylethanolamine was 3.3%, indicating that the method had good repeatability.

[0042] Embodiment 5: Spiked recovery rate investigation: accurately weigh 1000 mg of the progesterone soft capsule content, place it in a 10 mL volumetric flask, add chloroform-methanol solution (volume ratio 2:1) to dissolve soybean lecithin, accurately measure 1 mL of phosphatidylcholine standard stock solution and 0.5 mL of phosphatidylethanolamine standard stock solution, add them to the volumetric flask, dilute to the mark with chloroform-methanol solution (volume ratio 2:1), shake well, filter, discard 2 mL of the initial filtrate, and take the subsequent filtrate (prepare 6 portions in parallel). Samples were injected under the chromatographic conditions of Example 1 and Example 2, and the spiked recovery rate experimental results are shown in Table 4 below: Table 4: Results of the spike recovery test

[0043] It was determined that the spiked recovery rate of soybean phosphatidylcholine was between 84.38% and 116.16%, with an average recovery rate of 95.19%; the spiked recovery rate of phosphatidylethanolamine was between 88.96% and 106.08%, with an average recovery rate of 98.65%, which met the analysis requirements.

[0044] The above test results show that the method proposed by the present invention has good specificity, repeatability and accuracy for determining the contents of soybean phosphatidylcholine and phosphatidylethanolamine, two components of soybean lecithin in soft capsules, and is simple and easy to operate.

[0045] Comparative Example 1: The experimental conditions of this comparative example are: Chromatographic column model and specifications: Silica gel as filler (RD-SiO2 4.0*250mm, 5μm) 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); Flow rate: 1.0 mL / min; column temperature: 40 °C; Detector: evaporative light scattering detector, drift tube temperature: 72°C; carrier gas flow rate: 2.0L / min; Injection volume: 20 μL; The gradient elution program is shown in Table 5: Table 5: Gradient elution program of Comparative Example 1

[0046] Diluent (blank solution): chloroform-methanol (2:1); Test solution: Take an appropriate amount of soybean lecithin sample, add diluent to dissolve and dilute to make a solution of about 0.3 mg / mL; take the contents of progesterone soft capsule, add diluent to dissolve and dilute to make a solution of about 200 mg / mL.

[0047] Determination: When the instrument baseline is stable, take blank solution and test solution respectively, inject them into the chromatograph, and record the chromatogram. Figure 5 and Figure 6 As shown; HPLC-ELSD Figure 5 and Figure 6 Compared with the HPLC-UV spectra of Example 1 and Example 2, the baseline noise near the detected soybean phosphatidylcholine peak is large, and the phosphatidylethanolamine peak is interfered by the adjacent peaks, which affects the accuracy of content determination.

[0048] Comparative Example 2: The experimental conditions of this comparative example are: Chromatographic column model and specifications: Octadecylsilane bonded silica gel as filler (RD-C18 4.6*150mm, 3.5μm) Mobile phase: methanol-acetonitrile-water (50:40:10); Flow rate: 1.0 mL / min; column temperature: 35 °C; Detector: UV detector, detection wavelength: 206nm; injection volume: 20μL; Diluent (blank solution): chloroform-methanol (2:1); Positioning solution: Take 200 μg / mL soybean phosphatidylcholine standard solution and phosphatidylethanolamine standard solution as positioning solutions respectively; Mixed solution: Take solutions of soybean phosphatidylcholine and phosphatidylethanolamine with a concentration of 200 μg / mL respectively as a mixed solution; Test solution: Take the contents of progesterone soft capsules, add diluent to dissolve and dilute to make a solution of about 200 mg / mL.

[0049] Determination: When the instrument baseline is stable, take blank solution, positioning solution, mixed solution and test solution respectively, inject them into the chromatograph, and record the chromatogram. Figure 7 As shown; Under this method, no obvious peaks appeared for soybean phosphatidylcholine and phosphatidylethanolamine.

[0050] Comparative Example 3: The experimental conditions of this comparative example are: Chromatographic column model and specifications: Octadecylsilane bonded silica gel as filler (RD-C18 4.6*150mm, 3.5μm) Mobile phase: methanol-acetonitrile (90:10); Flow rate: 1.0 mL / min; column temperature: 35 °C; Detector: UV detector, detection wavelength: 206nm; injection volume: 20μL; Diluent (blank solution): chloroform-methanol (2:1); Positioning solution: Take 200 μg / mL soybean phosphatidylcholine standard solution and phosphatidylethanolamine standard solution as positioning solutions respectively; Mixed solution: Take solutions of soybean phosphatidylcholine and phosphatidylethanolamine with a concentration of 200 μg / mL respectively as a mixed solution; Determination: When the instrument baseline is stable, take blank solution, positioning solution and mixed solution respectively, inject them into the chromatograph, and record the chromatogram. Figure 8 As shown; Soybean phosphatidylcholine and phosphatidylethanolamine cannot be completely separated by this method.

[0051] Comparative Example 4: The experimental conditions of this comparative example are: Chromatographic column model and specifications: Octadecylsilane bonded silica gel as filler (XDB C18 4.6*150mm, 3.5μm) Mobile phase: methanol-acetonitrile-isopropanol (80:10:10); Flow rate: 1.0 mL / min; column temperature: 35 °C; Detector: UV detector, detection wavelength: 206nm; injection volume: 20μL; Diluent (blank solution): chloroform-methanol (2:1); Positioning solution: Take 200 μg / mL soybean phosphatidylcholine standard solution and phosphatidylethanolamine standard solution as positioning solutions respectively; Mixed solution: Take solutions of soybean phosphatidylcholine and phosphatidylethanolamine with a concentration of 200 μg / mL respectively as a mixed solution; Determination: When the instrument baseline is stable, take blank solution, positioning solution and mixed solution respectively, inject them into the chromatograph, and record the chromatogram. Fig. 9 As shown; Soybean phosphatidylcholine and phosphatidylethanolamine cannot be completely separated by this method.

[0052] Comparative Example 5: The experimental conditions of this comparative example are: Chromatographic column model and specifications: amino-bonded silica gel as filler (Aepax Hp-Amino 4.6*250mm, 5μm) Mobile phase: methanol-acetonitrile (40:60), methanol-acetonitrile (50:50), methanol-acetonitrile (55:45), methanol-acetonitrile (40:60); Flow rate: 1.0 mL / min; column temperature: 35 °C; Detector: UV detector, detection wavelength: 206nm; injection volume: 20μL; Diluent (blank solution): chloroform-methanol (2:1); Positioning solution: Take a 200 μg / mL phosphatidylethanolamine standard solution as the positioning solution; Determination: When the instrument baseline is stable, take blank solution and positioning solution respectively, inject them into the chromatograph, and record the chromatogram. Fig.10 As shown; Under these four mobile phase conditions on the amino column, phosphatidylethanolamine and solvent peaks could not be completely separated.

Claims

1. A method for determining the content of soybean phosphatidylcholine or phosphatidylethanolamine in soft capsules, characterized in that: The steps include: (1) Weigh soybean phosphatidylcholine standard and phosphatidylethanolamine standard respectively, dissolve them in solvent and make up to volume, and prepare soybean phosphatidylcholine standard solution and phosphatidylethanolamine standard solution with different concentration gradients respectively; (2) Weigh the sample to be tested from the contents of the soft capsule, dissolve it with a solvent and make it constant, filter it and take the filtrate to obtain a sample solution to be tested; (3) The standard solution and the sample solution to be tested are sampled and analyzed by a high performance liquid chromatograph-ultraviolet detector, the soybean phosphatidylcholine or phosphatidylethanolamine component is qualitatively analyzed according to the retention time of the standard, and the peak area of ​​the soybean phosphatidylcholine or phosphatidylethanolamine is determined; wherein: The chromatographic conditions for the analysis of soybean phosphatidylcholine by high performance liquid chromatography include: using a silica gel column, using methanol-water-glacial acetic acid-triethylamine as mobile phase A and n-hexane-isopropanol-mobile phase A as mobile phase B for isocratic elution, and then detecting with an ultraviolet detector; The chromatographic conditions for the sample injection analysis of phosphatidylethanolamine by high performance liquid chromatography include: using an amino-bonded silica gel column, using a methanol solution-acetonitrile as a mobile phase for isocratic elution, and then detecting with an ultraviolet detector; (4) Determine the standard solution with different concentration gradients obtained in step (1) according to the method of step (3), with the concentration of soybean phosphatidylcholine or phosphatidylethanolamine in the standard solution as the horizontal axis and the peak area of ​​the measured standard soybean phosphatidylcholine or phosphatidylethanolamine as the vertical axis, and obtain the standard curve and standard curve equation by linear regression; (5) Determine the sample solution obtained in step (2) according to the method of step (3), substitute the peak area of ​​the soybean phosphatidylcholine or phosphatidylethanolamine of the sample to be tested obtained into the standard curve equation, calculate the concentration of soybean phosphatidylcholine or phosphatidylethanolamine in the sample solution to be tested, and then calculate the mass fraction of soybean phosphatidylcholine or phosphatidylethanolamine in the sample to be tested, thereby achieving the determination of the soybean phosphatidylcholine or phosphatidylethanolamine content in the soft capsule.

2. The measuring method according to claim 1, characterized in that In steps (1) and (2), the solvent is a chloroform-methanol solution, wherein the volume ratio of chloroform to methanol is 1-4:

1.

3. The measuring method according to claim 2, characterized in that In step (1), the preparation method of preparing soybean phosphatidylcholine standard solution and phosphatidylethanolamine standard solution with different concentration gradients comprises the following steps: weighing soybean phosphatidylcholine standard and phosphatidylethanolamine standard respectively, dissolving them with chloroform-methanol solution to prepare standard stock solution, and then quantitatively diluting with the chloroform-methanol solution to prepare standard solution with different concentration gradients, wherein the concentration gradient of soybean phosphatidylcholine or phosphatidylethanolamine in the standard solution is 10 μg / mL~200 μg / mL.

4. The measuring method according to claim 1, characterized in that In step (2), the filter head used for filtration is a 0.45 μm nylon 66 filter head.

5. The measuring method according to claim 1, characterized in that In step (3), the chromatographic conditions for the analysis of soybean phosphatidylcholine by high performance liquid chromatography also include: the size of the silica gel column is 4.6 mm×250 mm, the column temperature is 35-45° C., the injection volume is 10-50 μL; and the wavelength of the ultraviolet detector is 205 nm.

6. The measuring method according to claim 1, characterized in that In step (3), in the mobile phase A, the volume ratio of methanol, water, glacial acetic acid and triethylamine is 80-90:10-20:0.45:0.05; in the mobile phase B, the volume ratio of n-hexane, isopropanol and mobile phase A is 17-23:45-51:32; the volume ratio of mobile phase A to mobile phase B is (30-50):(70-50), and the flow rate is 0.5-2 mL / min.

7. The measuring method according to claim 1, characterized in that In step (3), the chromatographic conditions for the analysis of phosphatidylethanolamine by high performance liquid chromatography also include: the specifications of the amino-bonded silica gel column are 4.6 mm×250 mm, the column temperature is 35-45° C., and the injection volume is 20-100 μL.

8. The measuring method according to claim 1, characterized in that In step (3), the methanol solution is obtained by mixing glacial acetic acid, triethylamine and methanol in a volume ratio of 0.40-0.50:0.05:100; in the methanol solution-acetonitrile, the volume ratio of methanol solution to acetonitrile is (15-35): (85-65), and the flow rate is 0.5-2 mL / min.

9. The assay method according to claim 1, characterized in that In step (5), the concentration of soybean phosphatidylcholine in the standard solution X1 is taken as the horizontal coordinate, the peak area of ​​phosphatidylcholine Y1 is taken as the vertical coordinate, and the linear regression equation of the standard curve is obtained: Y1=4872.287 X1-4919.591, and the correlation coefficient r1 is 0.999; the concentration of phosphatidylethanolamine in the standard solution X2 is taken as the horizontal coordinate, the peak area of ​​phosphatidylethanolamine Y2 is taken as the vertical coordinate, and the linear regression equation of the standard curve is obtained: Y2=35111.400 X2+75330.667, and the correlation coefficient r2 is 0.

999.

10. The assay method according to any one of claims 1 to 9, characterized in that: In step (5), the mass fraction of soybean phosphatidylcholine or phosphatidylethanolamine in the sample to be tested is calculated as M= ; Wherein, M is the content of soybean phosphatidylcholine or phosphatidylethanolamine in the sample to be tested, unit: %; c is the concentration of soybean phosphatidylcholine or phosphatidylethanolamine in the sample solution to be tested calculated in step (5), unit: μg / mL; V is the total volume of the sample solution to be tested prepared in step (2), unit: mL; m is the mass of the sample to be tested weighed in step (2), unit: mg.

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