Vitamin K1 micelle encapsulation efficiency determination method

Abstract

The invention relates to the technical field of pharmaceutical analysis, and particularly discloses a method for measuring the encapsulation efficiency of vitamin K1 micelles, which comprises the following steps: detecting the content of free vitamin K1 and the content of total vitamin K1 by high performance liquid chromatography, taking an aqueous solution containing 0.1% of trifluoroacetic acid as a mobile phase A, taking ethanol as a mobile phase B, and carrying out gradient elution by a C18 chromatographic column to obtain the encapsulation efficiency of the vitamin K1 micelles. The elution procedure comprises the following steps: starting elution from 60% of a mobile phase A and 40% of a mobile phase B, gradually reducing the proportion of the mobile phase A, gradually increasing the proportion of the mobile phase B, adjusting the proportion of the mobile phase A to be 20% and the proportion of the mobile phase B to be 80% in 13 minutes, increasing the proportion of the mobile phase A, reducing the proportion of the mobile phase B, adjusting the proportion of the mobile phase A to be 60% and the proportion of the mobile phase B to be 40% in 14 minutes, and continuing elution for 6 minutes; proper wavelength and column temperature are set, and the sample injection volume is 5 microliters. The detection method provided by the invention can effectively detect the encapsulation efficiency of the vitamin K1 micelle injection, and is simple to operate and stable and reliable in result.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the micelle encapsulation efficiency of vitamin K1. Background Technology

[0002] Vitamin K1 (VK1) is a fat-soluble vitamin and an essential cofactor for the synthesis of prothrombin from clotting factors II, VII, IX, and X in the liver. It is used to treat hypoprothrombinemia, vitamin K deficiency, and to prevent and treat neonatal hemorrhagic diseases. Mixed micelles composed of bile salts and phospholipids serve as a novel drug delivery system with high biocompatibility, improving the solubility of poorly soluble drugs while reducing the risk of allergic reactions. However, studies have reported adverse reactions with VK1 micelle solutions in clinical use, possibly caused by free VK1 in the injection solution. Therefore, the encapsulation efficiency of vitamin K1 micelle injections is a key indicator affecting drug safety. The composition of vitamin K1 micelles includes: the active ingredient is vitamin K1, and other components are soybean lecithin, glycinecholic acid, sodium hydroxide, hydrochloric acid, and water for injection.

[0003] The only reported invention patent for determining the encapsulation efficiency of vitamin K1 injection is publication number CN119064492A published by Shijiazhuang No. 4 Pharmaceutical Co., Ltd. This method uses a non-polar macroporous adsorption resin, mixes the solution, and centrifuges it to separate the vitamin K1 injection solution into three layers from top to bottom: a "viscous droplet" layer, a micelle layer, and a resin layer. The contents of the micelle and resin layers are then tested using high-performance liquid chromatography (HPLC), and the encapsulation efficiency is calculated. However, this method is cumbersome, time-consuming, and has a low recovery rate. Therefore, a new method for determining the micelle encapsulation efficiency of vitamin K1 is provided. Summary of the Invention

[0004] The purpose of this invention is to address the deficiencies of the prior art by providing a method for determining the encapsulation efficiency of vitamin K1 micelles, thereby solving the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for determining the encapsulation efficiency of vitamin K1 micelles, wherein the content of free vitamin K1 and total vitamin K1 is detected by high performance liquid chromatography (HPLC). An aqueous solution containing 0.1% trifluoroacetic acid is used as mobile phase A, and ethanol is used as mobile phase B. Gradient elution is performed using a C18 column. The elution program is as follows: elution begins with 60% mobile phase A and 40% mobile phase B; the proportion of mobile phase A is gradually decreased, and the proportion of mobile phase B is gradually increased until, after 13 minutes, mobile phase A reaches 20% and mobile phase B reaches 80%; the proportion of mobile phase A is then increased, and the proportion of mobile phase B is decreased; at 14 minutes, mobile phase A is adjusted to 60% and mobile phase B to 40%; elution continues for 6 minutes. Appropriate wavelength and column temperature are set, and the injection volume is 5 μl.

[0006] The specific steps are as follows:

[0007] Step 1: Select anhydrous ethanol as the solvent;

[0008] Step 2, Test Solution: Take 200 μl of the filtrate obtained by filtering this product through a 0.45 μm nylon membrane, place it in a 25 mL brown volumetric flask, add 10 mL of anhydrous ethanol, sonicate to dissolve, and then dilute to volume with 90% ethanol to obtain the filtered free vitamin K1 test solution; take 200 μl of this product before filtration and prepare it in the same way to obtain the total vitamin K1 test solution before filtration.

[0009] Step 3, Reference solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing about 100 μg per ml;

[0010] Step 4: Take the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatograms, and measure the free vitamin K1 content and total vitamin K1 content with the reference standard. Record them as: free VK1 content and total VK1 content in the sample, respectively.

[0011] Step 5, Calculation formula:

[0012]

[0013] As a preferred embodiment of the present invention, the flow rate of the high performance liquid chromatography is 0.9 ml / min to 1.1 ml / min.

[0014] As a preferred embodiment of the present invention, the column temperature of the high performance liquid chromatography is 25-35°C.

[0015] As a preferred embodiment of the present invention, the column temperature of the high-performance liquid chromatography is 30°C.

[0016] As a preferred embodiment of the present invention, the wavelength of the high-performance liquid chromatography method is 252nm to 256nm.

[0017] As a preferred embodiment of the present invention, the wavelength of the high-performance liquid chromatography is 254 nm.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] The detection method of the present invention can effectively detect the encapsulation rate of vitamin K1 micelle injection. It is simple to operate and the results are stable and reliable, providing effective assurance for the quality of vitamin K1 micelle injection products. It has positive effects and practical application value.

[0020] The analytical method disclosed in this invention can effectively detect the encapsulation efficiency in vitamin K1 micelles. This method exhibits good specificity, high sensitivity, linearity, precision (injection precision, repeatability, intermediate precision), robustness, and is simple to operate, providing reliable results. Therefore, it can be used to control the encapsulation efficiency in vitamin K1 micelles, effectively ensuring the quality of vitamin K1 micelle products. Attached Figure Description

[0021] Figure 1 It involves examining the liquid chromatogram of a blank excipient solution (containing soybean lecithin and glycocholic acid);

[0022] Figure 2 This is the liquid chromatogram of the reference solution;

[0023] Figure 3 This is the liquid chromatogram of the test sample solution;

[0024] Figure 4 This is a linear graph of vitamin K1 in this invention. Detailed Implementation

[0025] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0026] Example: Please refer to Figure 1-3 This invention provides a technical solution: a method for determining the encapsulation efficiency of vitamin K1 micelles.

[0027] I. Encapsulation efficiency testing methods

[0028] Studies have reported adverse reactions even with the clinical application of vitamin K1 micelle solutions, possibly caused by free vitamin K1 in the injection. Therefore, the encapsulation efficiency of vitamin K1 micelle injections is a key indicator affecting drug safety.

[0029] This invention utilizes a filtration method and high-performance liquid chromatography (HPLC) to determine the peak area of ​​free vitamin K1, thereby determining its concentration and calculating the encapsulation efficiency. The proposed detection method is as follows:

[0030] (1) Solvent: Anhydrous ethanol

[0031] (2) Excipient solution: Take about 15 mg of soybean lecithin and about 10 mg of glycinecholic acid, put them in a 100 ml volumetric flask, add solvent to dilute to the mark, and shake well.

[0032] (3) Test solution: Take 200 μl of the filtrate obtained by filtering this product through a 0.45 μm nylon filter membrane, place it in a 25 mL brown volumetric flask, add 10 mL of anhydrous ethanol, dissolve by sonication, and then dilute to volume with 90% ethanol to obtain the free VK1 test solution (after filtration); take 200 μl of this product before filtration and prepare it in the same way to obtain the total VK1 test solution (before filtration).

[0033] (4) Reference solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 100 μg per ml.

[0034] (5) Chromatographic conditions: Octadecyl bonded silica gel was used as the stationary phase (4.6 mm × 150 mm, 5 μm); mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid and mobile phase B was ethanol. Gradient elution was performed according to the program in the table below, with a flow rate of 1.0 ml / min; the detection wavelength was 254 nm; and the injection volume was 5 μl.

[0035] The gradient elution procedure is shown in the table below:

[0036] t(min) A(％) B(％) 0 60 40 13 20 80 14 60 40 20 60 40

[0037] (5) System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number, calculated based on the vitamin K1 peak, shall not be less than 3000.

[0038] (6) Accurately measure the excipient solution, reference solution, and test sample and inject them directly; the chromatogram is as follows: Figure 1 and Figure 2 and Figure 3 As shown.

[0039] Conclusion: The excipient solution did not interfere with the elution of vitamin K1; the retention time of the main peak in the test solution was consistent with that in the reference solution. This method is tentatively designated as the determination method for the encapsulation efficiency of vitamin K1 micelles, and methodological validation will be conducted.

[0040] II. Encapsulation efficiency methodology research and evaluation

[0041] 1. Instruments and conditions: High performance liquid chromatography was used with octadecyl bonded silica gel as the stationary phase (4.6 mm × 150 mm, 5 μm); mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was ethanol. Gradient elution was performed according to the program in the table below, with a flow rate of 1.0 ml / min; the detection wavelength was 254 nm; and the injection volume was 5 μl.

[0042] The gradient elution procedure is shown in the table below:

[0043] t(min) A(％) B(％) 0 60 40 13 20 80 14 60 40 20 60 40

[0044] 2. Experimental steps:

[0045] 2.1 Specificity

[0046] (1) Solvent: Anhydrous ethanol

[0047] (2) Excipient solution: Take about 15 mg of soybean lecithin and about 10 mg of glycinecholic acid, put them in a 100 ml volumetric flask, add solvent to dilute to the mark, and shake well.

[0048] (3) Test solution: Take 200 μl of this product, place it in a 25 mL brown volumetric flask, add 10 mL of anhydrous ethanol, sonicate to dissolve, and then dilute to volume with 90% ethanol to obtain the solution.

[0049] (4) Reference solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 100 μg per ml.

[0050] Under the above chromatographic conditions, accurately measure 20 μl each of the blank solvent, blank excipient solution, mixed solution, each impurity localization solution, and test solution, and inject them into the liquid chromatograph, recording the chromatograms. The results are shown in Table 1 below.

[0051] Table 1: Results of Specificity Tests

[0052]

[0053] Conclusion: The retention time of the main peak in the reference solution was consistent with that in the test solution. The blank solvent and blank excipient solution did not interfere with the vitamin K1 elution peak, meeting the validation requirements. This method exhibits good specificity.

[0054] 2.2 Linearity and Range

[0055] (1) Solvent: Anhydrous ethanol

[0056] (2) Linear stock solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing about 1 mg per 1 ml, which is used as the linear stock solution.

[0057] (3) Linear solutions: Accurately measure 1.0 ml, 2.0 ml, 3.0 ml, 5.0 ml, 10 ml, and 3 ml of linear stock solution into 100 ml, 100 ml, 100 ml, 100 ml, 100 ml, and 20 ml volumetric flasks, respectively, dilute with solvent to the mark, and shake well to obtain standard solutions 1 to 6.

[0058] Linear solutions of each concentration gradient were injected into the liquid chromatograph, and the chromatograms were recorded. A linear regression was performed with the natural logarithm of the injection concentration C (ug / ml) on the x-axis and the natural logarithm of the peak area A on the y-axis. The experimental results are shown in Table 2 below.

[0059] Table 2: Results of Vitamin K1 Linearity and Range Tests

[0060]

[0061] Conclusion: Vitamin K1 showed a good linear relationship with a correlation coefficient r greater than 0.999 in the concentration range of approximately 10 μg / ml to 100 μg / ml.

[0062] 2.3 Precision

[0063] 2.3.1 Sample injection precision

[0064] (1) Solvent: Anhydrous ethanol

[0065] (2) Reference solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 100 μg per ml.

[0066] Accurately measure the reference solution and inject it into the liquid chromatograph. Inject six times consecutively, record the chromatogram, and calculate the peak area RSD. The injection precision results are shown in Table 3 below.

[0067] Table 3: Results of the injection precision test (n=6)

[0068]

[0069] Conclusion: When the same reference solution was injected six times consecutively, the RSD of the vitamin K1 peak area was less than 2%, indicating that the injection precision of this method was good.

[0070] 2.3.2 Repeatability

[0071] (1) Solvent: Anhydrous ethanol

[0072] (2) Reference solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 100 μg per ml.

[0073] (3) Test solution: Take 200 μl of the filtrate obtained by filtering this product through a 0.45 μm nylon filter membrane, place it in a 25 mL brown volumetric flask, add 10 mL of anhydrous ethanol, sonicate to dissolve, and then dilute to volume with 90% ethanol to obtain the free VK1 test solution (after filtration); take 200 μl of this product before filtration and prepare it in the same way to obtain the total VK1 test solution (before filtration).

[0074] Inject the blank solvent, reference solution, and test solution into the liquid chromatograph, record the chromatograms, and calculate the encapsulation efficiency and relative standard deviation (RSD) based on the peak area using the external standard method. The test results are shown in the table below.

[0075] Table 4: Repeatability Test Results (Test Solution)

[0076]

[0077] Conclusion: The RSD of the nitrosamine impurity content determination results in the 6 test sample solutions was less than 10%, and the repeatability of this method was good.

[0078] 2.3.3 Intermediate Precision

[0079] The nitrosamine impurity content of the test solution was determined at different times by different testers using different instruments according to the method described in the repeatability test section. The test results are shown in the table below.

[0080] Table 5: Intermediate precision results (test solution)

[0081]

[0082] Conclusion: The RSD of the nitrosamine impurity content determination results in the 12 test sample solutions was less than 5%, indicating that the intermediate precision of this method is good.

[0083] 2.4 Accuracy

[0084] (1) Solvent: Anhydrous ethanol

[0085] (2) Reference solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 100 μg per ml.

[0086] (3) Test solution (filtrate): Assemble a filter using a replaceable membrane filter and a 0.45μm nylon filter membrane. Accurately transfer 1ml of the known prescription drug solution into the above filter and filter it completely through the 0.45μm nylon filter membrane. Collect the filtrate and transfer it completely to a 100ml volumetric flask with anhydrous ethanol. Dilute to the mark with anhydrous ethanol and shake well.

[0087] (4) Test solution (filter): Elute the filter membrane and filter chamber with anhydrous ethanol to dissolve and transfer the residual VK1 to a 100ml volumetric flask, dilute to the mark with anhydrous ethanol, and shake well.

[0088] Take the above reference solution and test solution, inject them into the liquid chromatograph, and calculate the recovery rate. See Table 6 below for details.

[0089] Table 6: Accuracy Results

[0090]

[0091] As shown in Table 6 above, the recovery rate of this method is between 98.7% and 100.4%, which meets the validation requirements (95% to 105%). This confirms that the method has good accuracy.

[0092] 2.5 Durability

[0093] (1) Solvent: Anhydrous ethanol

[0094] (2) Reference solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 100 μg per ml.

[0095] (3) Test solution: Take 200 μl of the filtrate obtained by filtering this product through a 0.45 μm nylon filter membrane, place it in a 25 mL brown volumetric flask, add 10 mL of anhydrous ethanol, dissolve by sonication, and then dilute to volume with 90% ethanol to obtain the free VK1 test solution (after filtration); take 200 μl of this product before filtration and prepare it in the same way to obtain the total VK1 test solution (before filtration).

[0096] The detection conditions were appropriately varied: flow rate 1.0 ml / min ± 0.1 ml / min, column temperature (30℃ ± 5℃), detection wavelength (254 nm ± 2 nm), and different batches of chromatographic columns. The changes in the content determination results of lysophospholipids in the test sample solution under different conditions were investigated. The experimental condition variation parameters and results are shown in Tables 7 and 8 below.

[0097] Table 7: Parameters for variation of chromatographic conditions in durability testing

[0098]

[0099] Table 8: Durability Test Results

[0100]

[0101]

[0102] Conclusion: The method demonstrates good robustness when the difference in encapsulation efficiency of the test sample under varying chromatographic conditions is less than 2%.

[0103] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for determining the encapsulation efficiency of vitamin K1 micelles, characterized in that: The content of free vitamin K1 and total vitamin K1 was determined by high performance liquid chromatography (HPLC). Aqueous solution containing 0.1% trifluoroacetic acid was used as mobile phase A, and ethanol as mobile phase B. Gradient elution was performed using a C18 column. The elution program was as follows: elution began with 60% mobile phase A and 40% mobile phase B, gradually decreasing the proportion of mobile phase A and gradually increasing the proportion of mobile phase B until, after 13 minutes, mobile phase A reached 20% and mobile phase B reached 80%. Then, the proportion of mobile phase A was increased and the proportion of mobile phase B decreased until, after 14 minutes, mobile phase A was adjusted to 60% and mobile phase B to 40%, and elution continued for 6 minutes. Appropriate wavelength and column temperature were set, and the injection volume was 5 μl. The specific steps are as follows: Step 1: Select anhydrous ethanol as the solvent; Step 2, Test Solution: Take 200 μl of the filtrate obtained by filtering this product through a 0.45 μm nylon membrane, place it in a 25 mL brown volumetric flask, add 10 mL of anhydrous ethanol, sonicate to dissolve, and then dilute to volume with 90% ethanol to obtain the filtered free vitamin K1 test solution; take 200 μl of this product before filtration and prepare it in the same way to obtain the total vitamin K1 test solution before filtration. Step 3, Reference solution: Take an appropriate amount of vitamin K1 reference standard, accurately weigh it, dissolve it in solvent and quantitatively dilute it to prepare a solution containing about 100 μg per ml; Step 4: Take the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatograms, and measure the free vitamin K1 content and total vitamin K1 content with the reference standard. Record them as: free VK1 content and total VK1 content in the sample, respectively. Step 5, Calculation formula:

2. The method for determining the encapsulation efficiency of vitamin K1 micelles according to claim 1, characterized in that: The flow rate of the high-performance liquid chromatography method is 0.9 ml / min to 1.1 ml / min.

3. The method for determining the encapsulation efficiency of vitamin K1 micelles according to claim 1, characterized in that: The column temperature for the high-performance liquid chromatography method is 25–35 °C.

4. The method for determining the encapsulation efficiency of vitamin K1 micelles according to claim 3, characterized in that: The column temperature for the high-performance liquid chromatography method is 30℃.

5. The method for determining the encapsulation efficiency of vitamin K1 micelles according to claim 1, characterized in that: The wavelength of the high-performance liquid chromatography method is 252 nm to 256 nm.

6. The method for determining the encapsulation efficiency of vitamin K1 micelles according to claim 5, characterized in that: The wavelength of the high-performance liquid chromatography method is 254 nm.

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