Method for determining entrapment rate of irinotecan liposome injection

Abstract

The invention provides a method for determining the entrapment rate of an irinotecan liposome injection. The method comprises the following steps: 1) separating the irinotecan liposome injection into an entrapment body solution and a free body solution; (2) preparing a test solution A from the encapsulation body solution, and preparing a test solution B from the free body solution, wherein both the steps comprise a heating step; and (3) respectively testing the irinotecan concentrations in the test solution A and the test solution B through high performance liquid chromatography. According to the method disclosed by the invention, the encapsulation efficiency of the irinotecan liposome injection can be detected only through high performance liquid chromatography, and the problem that a test solution is unstable is solved.

Description

Technical Field

[0001] The invention belongs to the field of small molecule compound synthesis, and specifically relates to a method for preparing an imidazole derivative. Background Art

[0002] Irinotecan hydrochloride is a semi-synthetic water-soluble camptothecin derivative and an anti-tumor drug launched in 1994. Irinotecan hydrochloride has good water solubility, higher anti-tumor activity and lower toxicity than camptothecin. It is still one of the main treatments for metastatic colon cancer and rectal cancer. Its approved dosage form is injection, and the main adverse reactions are neutropenia and diarrhea. Long-term high-dose use is difficult for patients to tolerate. Making it into a liposome injection can not only improve adverse reactions, but also increase the stability of the drug. Currently, liposome injection is the most widely used dosage form.

[0003] The encapsulation rate of liposomes refers to the percentage of the encapsulated substance (such as a drug) in the total amount of the drug in the liposome suspension. It is an important indicator of liposome quality control, reflecting the degree to which the drug is encapsulated by the carrier. The percentage encapsulation rate is: EN% = (1-C f / C t )×100%, where Cf is the amount of free drug and Ct is the total amount of drug in the liposome suspension. Therefore, the encapsulation efficiency is usually determined by separating the free drug and liposomes in the preparation, measuring them separately, and then calculating the encapsulation efficiency. It is usually required that the drug encapsulation efficiency of liposomes is more than 80%.

[0004] There are many reports on the determination method of liposome encapsulation rate in the prior art. Zhao Ligang et al. (Determination of encapsulation rate and efficacy of long-circulating vinorelbine tartrate liposomes, Journal of China Medical University, 2010, 41(6): pp508-512) determined the encapsulation rate by ultrafiltration, but the mechanical force during centrifugation may cause particle aggregation, resulting in the destruction of liposomes, and the ultrafiltration membrane may adsorb drugs, resulting in measurement errors, and the "concentration polarization" phenomenon also limits its use. Ultrafiltration instruments have high requirements and are expensive. In addition, high-speed centrifugation has a poor separation effect on liposomes with smaller particle sizes (<100nm), and the liposomes cannot be completely precipitated. Huang Junqin et al. (Preparation of Gemcitabine Hydrochloride Liposomes and Determination of Content and Encapsulation Rate, Chinese Journal of Antibiotics, January 2010, Vol. 35, No. 1, pp30-32) adopted dialysis method to determine the encapsulation rate, but the drug encapsulated in the liposome may leak out, and this method is not applicable to poorly soluble drugs and is time-consuming. Zhang Jianjian et al. (Preparation of Irinotecan Hydrochloride Liposomes and Study on In Vitro Drug Release Characteristics, Chinese Pharmacy, Vol. 19, No. 10, 2008, pp762-764) determined the encapsulation rate by gel chromatography-ultraviolet spectroscopy, but the ultraviolet determination method may be interfered by excipients and impurities, and the specificity is not as good as that of high performance liquid chromatography.

[0005] Zhang Xiaolei et al. (Study on the Determination Method of Encapsulation Efficiency of Compound Irinotecan Hydrochloride and Fluorouracil Liposome Injection, Chinese Modern Applied Pharmacy, Vol. 38, No. 13, July 2021, pp1588-1593) determined the encapsulation efficiency by gel chromatography. The method adopted gel column chromatography, pH 6.5 phosphate buffer solution as eluent, under the established HPLC-DAD chromatographic conditions, using Kromasil C18 column (250mm×4.6mm, 5μm), methanol-pH 6.6 phosphate buffer (5∶95) as mobile phase A, methanol-pH 6.6 phosphate buffer (60∶40) as mobile phase B, gradient elution. Column temperature 35℃, flow rate 1.0mL·min-1. The detection wavelengths were 255 and 268nm, respectively. CN116718696A discloses a method for measuring the encapsulation rate of liposome injection, which uses Sepharose CL-4B agarose gel chromatography to separate encapsulated irinotecan and free irinotecan. According to this method and adjusted according to actual conditions, the peak area of ​​irinotecan in the test solution B measured by the encapsulation rate is unstable, and the possible reason is that there is a process of conversion of the intramolecular lactone to the carboxylic acid type structure of irinotecan. Both methods use high performance liquid chromatography, but the applicant finds that stable test results cannot be obtained under the measurement conditions, which is specifically manifested in that, when other conditions remain unchanged, the measured results also change with the change of the test solution placement time. This may be related to the instability of irinotecan under the test solution conditions. Summary of the invention

[0006] One of the purposes of the present invention is to solve the problem of unstable measurement results when analyzing the encapsulation rate of irinotecan liposome injection by liquid chromatography in the prior art.

[0007] According to the present invention, a method for determining the encapsulation efficiency of irinotecan liposome injection is provided, which comprises the following steps:

[0008] 1) separating the irinotecan liposome injection into an encapsulated body solution and a free body solution;

[0009] 2) preparing a test solution A from the encapsulated body solution and preparing a test solution B from the free body solution, both of which include a heating step;

[0010] 3) The irinotecan concentration C in the test solution A and the test solution B was tested by high performance liquid chromatography. A and C B , the encapsulation efficiency was calculated according to the following formula:

[0011] Encapsulation efficiency (%) = C A / (C A +C B)*100%.

[0012] In a specific embodiment of the present invention, the encapsulated body and the free body are separated by gel column chromatography, using agarose as the chromatography column material and 0.9% sodium chloride solution as the eluent.

[0013] In a specific embodiment of the present invention, the preparation of test solution A and test solution B includes:

[0014] 2-1) using 0.9% sodium chloride solution as an eluent, diluting the encapsulated body solution and the free body solution obtained in step 1) with a diluent to obtain diluents, respectively, wherein the diluent is one or more of methanol, isopropanol, acetonitrile and ethanol, wherein isopropanol is preferred;

[0015] 2-2) ultrasonically treating the dilution for 3-10 minutes, preferably 4-5 minutes;

[0016] 2-3) The diluted solution is formulated into a solution with a concentration of 0.04 to 12.9 μg / ml using a first solvent to obtain a test solution A and a test solution B, respectively, wherein the first solution is a mixed aqueous solution of sodium dihydrogen phosphate and 1-octane sodium sulfonate at pH 3.6, preferably, the concentration of sodium dihydrogen phosphate in the first solution is 2.8 g / L, and the concentration of sodium 1-octane sodium sulfonate is 1.8 g / L.

[0017] Preferably, when preparing the test solution A, the amount of the diluent is 0.7-2 times, preferably 1-1.5 times, the volume of the encapsulation solution, and the volume ratio of the diluent to the first solvent is 1:3-4.

[0018] Preferably, when preparing the test solution B, the amount of the diluent used is 0.5-1 times the volume of the free body solution, and the volume ratio of the diluent to the first solvent is 1:2-3.

[0019] Preferably, the heating temperature in step 2) is between 45-60° C. and the heating time is controlled between 30-50 minutes.

[0020] In a specific embodiment of the present invention, the following analysis conditions are used:

[0021] The chromatographic column is an octadecylsilane bonded silica gel column;

[0022] The mobile phase is the first solution-acetonitrile-methanol, the ratio is 40-60:10-20:15-25, wherein the preferred ratio is 53:20:27;

[0023] Column temperature: 35-45°C, preferably 40°C;

[0024] Flow rate: 0.8-1.2 ml / min, preferably 1.0 ml / min;

[0025] The detector is: an ultraviolet detector, with a detection wavelength of 235 to 255 nm, preferably 255 nm;

[0026] Injection volume: 20μl.

[0027] By adopting the method of the present invention, the encapsulation rate of irinotecan liposome injection can be detected only by high performance liquid chromatography, and the problem of instability of the test solution is overcome. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Hereinafter, some specific embodiments of the present invention will be described in detail in an exemplary and non-limiting manner with reference to the accompanying drawings. The same reference numerals in the accompanying drawings indicate the same or similar components or parts. It should be understood by those skilled in the art that these drawings are not necessarily drawn to scale. In the accompanying drawings:

[0029] Figure 1 The chromatograms of the test solution B at five different storage times in Example 1 are shown;

[0030] Figure 2 This is the detection chromatogram of Comparative Example 1 for test solution B at 5 different storage times. DETAILED DESCRIPTION

[0031] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and drawings. Obviously, the described embodiments are only exemplary, not all implementation methods. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0032] One of the characteristics of the present invention is that the test solution is heated before being subjected to high-performance liquid chromatography analysis. The inventors have found that the test solution after heating is stable under the test conditions and no concentration change is detected with the change of storage time.

[0033] The method according to the present invention comprises the following steps:

[0034] 1) separating the irinotecan liposome injection into an encapsulated body solution and a free body solution;

[0035] 2) preparing a test solution A from the encapsulated body solution and preparing a test solution B from the free body solution, both of which include a heating step;

[0036] 3) Test the irinotecan concentration C in test solution A and test solution B respectively A and C B , the encapsulation efficiency was calculated according to the following formula:

[0037] Encapsulation efficiency (%) = C A / (C A +C B )*100%.

[0038] Preparation of irinotecan liposome injection

[0039] In the present invention, irinotecan liposomes can be prepared by a method suitable for industrial implementation in the prior art, such as a thin film dispersion method, an ammonium sulfate gradient method, an ether injection method, and a reverse phase evaporation method, wherein the ammonium sulfate gradient method is preferred. In this method, a prescribed amount of cholesterol and cultured phosphatidylethanolamine are ultrasonically dissolved with an appropriate amount of ethanol, and the ethanol is removed by reduced pressure evaporation. An appropriate amount of ammonium sulfate aqueous solution is added, and the membrane is completely hydrated using a rotary evaporator. The hydrated mixture is circulated through an ultrafiltration system to remove the ammonium sulfate in the aqueous phase outside the liposome and replace it with an aqueous solution. The raw material drug aqueous solution is added to a water bath for heating and incubation, and then filtered with a microporous filter membrane.

[0040] In a typical embodiment of the present invention, the irinotecan hydrochloride liposome injection used is self-made by the applicant's company.

[0041] Separation of encapsulated and free bodies

[0042] In the present invention, the encapsulated irinotecan (i.e., the encapsulated body) and the unencapsulated part (the free body) in the preparation are first separated, and the preferred separation method is gel column chromatography. In order to prepare the chromatography column, an appropriate amount of gel can be taken, soaked and washed with deionized water, the upper aqueous solution is discarded, and the chromatography column tube is slowly loaded to a certain height, and then balanced with an eluent before use.

[0043] The chromatographic column material used for separation can be one of agarose or its derivatives, and dextran gel. In a specific embodiment, an agarose gel column is used, and a 0.9% sodium chloride solution is used as an eluent to separate the encapsulated body and the free body. During elution, the encapsulated body solution is first obtained, and then the free body solution is obtained by continuing the elution.

[0044] In another specific embodiment, the chromatography column is prepared using G-50 Sephadex.

[0045] Preparation of test solution A

[0046] Next, the obtained encapsulated body solution is prepared into a test solution A, and the solvent in the encapsulated body solution is the eluent used in the previous step, that is, 0.9% sodium chloride solution, and the amount used is the elution amount. In a typical embodiment, in order to separate the liposome injection, 50-100 times the volume of the eluent can be used to elute and obtain the encapsulated body solution. The solution is appropriately diluted with a diluent, and the diluent is one or more of methanol, isopropanol, acetonitrile and ethanol, wherein isopropanol is preferred. Isopropanol can destroy the structure of the liposome, so that the encapsulated irinotecan is free and can be detected by the detector. The amount of diluent can be 0.7-2 times the volume of the encapsulated body solution, preferably 1-1.5 times. The diluent is then preferably ultrasonically treated for 3-10 minutes, more preferably 4-5 minutes, and the purpose of ultrasonic treatment is to further promote demulsification.

[0047] Next, the solution is formulated into a predetermined concentration range with a first solvent so that the irinotecan in the test solution A falls within a detectable linear range. The irinotecan concentration in the test solution A in the present invention is preferably in the range of 0.04 to 12.9 μg / ml. The first solution is an aqueous solution containing sodium dihydrogen phosphate and 1-octane sodium sulfonate at a pH of 3.6. In a typical embodiment, the concentration of sodium dihydrogen phosphate in the first solution is 2.0-4.0 g / L, preferably 2.8 g / L, and the concentration of 1-octane sodium sulfonate is 1.0-3.0 g / L, preferably 1.8 g / L. The volume ratio of the diluent and the first solvent dilution can be in the range of 1:3 to 4.

[0048] The test solution A prepared by the above method is not only suitable for testing under the high performance liquid chromatography conditions of the present invention, but also can be well stabilized after being heated. In a typical embodiment of the present invention, water bath heating is adopted, and the water bath temperature is set between 45-60°C, preferably between 50-55°C. The time is controlled between 30-50 minutes, preferably between 30-40 minutes.

[0049] Preparation of test solution B

[0050] Next, the obtained free body solution is prepared into a test solution B, the solvent in the solution is a 0.9% sodium chloride solution, and the amount used is the elution amount. In a typical embodiment, the separated liposome injection can be eluted with 50-100 times the volume of the eluent to obtain a free body solution. The solution is appropriately diluted with a diluent, and the diluent is one or more of methanol, isopropanol, acetonitrile and ethanol, wherein isopropanol is preferred. The amount of diluent can be 0.5-1 times the volume of the free body solution. Then, the solution is formulated into a predetermined concentration range with the above-mentioned first solvent, so that the irinotecan in the test solution B falls into the detectable linear range. The volume ratio of the diluent and the first solvent dilution can be in the range of 1:2 to 3.

[0051] The test solution B prepared by the above method is not only suitable for testing under the high performance liquid chromatography conditions of the present invention, but also can be well stabilized after being heated. In a typical embodiment of the present invention, water bath heating is adopted, the water bath temperature is set between 45-60°C, and the time is controlled between 30-50 minutes.

[0052] Preparation of reference solution

[0053] Take irinotecan hydrochloride reference substance, dissolve it with methanol into 0.4-0.5 mg / ml solution, and obtain irinotecan stock solution. The stock solution is frozen for storage, and is further diluted with methanol into 0.04-12.9 μg / ml reference substance solution when used.

[0054] Liquid chromatography detection

[0055] The chromatographic conditions for detecting test solutions A and B are:

[0056] Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as filler;

[0057] Mobile phase: first solution - acetonitrile - methanol, the ratio is 40-60:10-20:15-25, preferably 53:20:27

[0058] Column temperature: 35-45°C, preferably 40°C;

[0059] Flow rate: 0.8-1.2 ml / min, preferably 1.0 ml / min;

[0060] The detector is: an ultraviolet detector, with a detection wavelength of 235 to 255 nm, preferably 255 nm;

[0061] Injection volume: 20μl.

[0062] Example 1

[0063] Preparation of separation column: Take 20ml of agarose gel CL-4B into a measuring cylinder, soak and stir in deionized water, let stand to separate, discard the upper aqueous solution, repeat the washing 4 to 5 times, slowly pour the washed agarose gel CL-4B aqueous solution into the chromatography column, and the column bed height is about 15cm. Rinse with deionized water, let stand for 10 minutes, open the drainage port at the bottom of the chromatography column, drain the liquid in the column, remove the excess agarose gel CL-4B to make the column bed height 12cm, and finally rinse the agarose gel CL-4B with 0.9% sodium chloride solution to balance.

[0064] Preparation of test solution A: Accurately measure 0.1 ml of irinotecan liposome injection into the prepared chromatography column, and add 0.25 ml of 0.9% sodium chloride solution after the sample completely enters the column bed. After the sample completely enters the column bed, add 7.75 ml of 0.9% sodium chloride solution. Use a 50 ml volumetric flask to collect the above solution, add 10 ml of isopropanol, ultrasonicate for 5 min, dilute to the scale with the first solution, heat in a 50°C water bath for 30 min, cool to room temperature, shake well, and use as test solution A.

[0065] Preparation of test solution B: After completing step b, add 7 ml of 0.9% sodium chloride solution, collect the above solution in a 20 ml volumetric flask, add 4 ml of isopropanol, ultrasonicate for 5 min, dilute to the scale with the first solution, place in a 50°C water bath and heat for 30 min, take out and cool to room temperature, shake well, and use this as test solution B.

[0066] Preparation of reference solution: Accurately weigh 43 mg of irinotecan hydrochloride reference substance and place it in a 100 ml volumetric flask. Add appropriate amount of methanol to dissolve and dilute to the mark to obtain irinotecan stock solution. Take the irinotecan stock solution and use methanol to prepare a 0.04-12.9 μg / ml reference solution.

[0067] Chromatographic analysis: The test solutions A and B were detected by high performance liquid chromatography, wherein the chromatographic conditions were as follows: a chromatographic column (4.6 mm × 250 mm, 5 μm) with octadecylsilane bonded silica as the filler, solution 1 (a mixed aqueous solution of sodium dihydrogen phosphate and 1-octane sodium sulfonate) - acetonitrile - methanol (53:20:27) as the mobile phase, a flow rate of 1.0 ml per minute; a column temperature of 40°C; a detection wavelength of 255 nm; and an injection volume of 20 μl.

[0068] The peak area of ​​the test solution B after being placed at room temperature for different time periods is measured. The test results are shown in Tables 1 and Figure 1 .

[0069] Table 1. Peak area test results of test solution B after different storage time:

[0070]

[0071] From Table 1 and Figure 1 It can be seen that the test solution prepared by the method of the present invention is stable under system conditions and room temperature, and no chemical changes occur.

[0072] Use test solution A and B respectively to do six parallel tests. The test results are shown in Table 2:

[0073] Table 2. Parallel test results of Example 1

[0074]

[0075] Example 2

[0076] The operation of Example 1 was repeated, except that the gel in step a was G-50 dextran gel, the column bed height was 11.5 cm, and the remaining operations were the same as in Example 1. Two parallel tests were performed, and the results are shown in Table 3.

[0077] Table 3. Experimental results of Example 2

[0078]

[0079] Comparative Example 1:

[0080] The step c of Example 1 was repeated to prepare the test solution B, except that the heating step was omitted. The peak area of ​​solution B after being placed at room temperature for different time periods was measured. The test results are shown in Tables 4 and Figure 2 .

[0081] Table 4. Peak area test results of test solution B at different storage times in comparative example 1

[0082]

[0083] From Table 4 and Figure 2 It can be seen that the test solution not prepared by the method of the present invention is unstable under storage conditions.

[0084] Example 3: Methodological Validation

[0085] 1. Injection precision test

[0086] Prepare reference stock solution, take an appropriate amount of irinotecan hydrochloride reference substance, accurately weigh, dissolve in methanol and dilute to make a solution containing about 0.43 mg of irinotecan per 1 ml. Dilute the reference stock solution with methanol to a reference solution of 0.04-12.9 μg / ml. Perform 6 parallel tests according to the liquid chromatography conditions in Example 1. The test results are shown in Table 5.

[0087] Table 5. Injection precision test results

[0088]

[0089]

[0090] The results showed that the method had good injection precision when 6 samples of reference solution were injected continuously, with the peak area RSD of 0.1% and retention time RSD of 0%.

[0091] 2. Linear relationship test

[0092] Reference substance stock solution: Take an appropriate amount of irinotecan hydrochloride reference substance, weigh accurately, dissolve in methanol and dilute to make a solution containing approximately 0.43 mg of irinotecan per 1 ml.

[0093] Reference substance solutions (1)-(7): The reference substance stock solutions were diluted with methanol to a reference substance solution of 0.04-12.9 μg / ml. The liquid chromatography conditions in Example 1 were respectively used for detection, and the chromatograms were recorded. The linear regression calculation was performed with the logarithm of the peak area as the ordinate and the logarithm of the concentration as the abscissa. The results are shown in Table 6.

[0094] Table 6. Linear relationship test results

[0095]

[0096] The results showed that in the range of 0.0443 μg / ml to 13.3044 μg / ml, the concentration and peak area showed a good linear relationship, the linear equation was y=67332.1100x+249.8059, and the correlation coefficient r was 1.000>0.999.

[0097] 3. Accuracy

[0098] Preparation of accuracy solution:

[0099] Accuracy Solution 1: Accurately measure 0.08 ml of irinotecan liposome injection into the prepared chromatography column, and prepare test solution A and test solution B according to "Example 1".

[0100] Accuracy Solution 2: Accurately measure 0.1 ml of irinotecan liposome injection into the prepared chromatography column, and prepare test solution A and test solution B according to "Example 1".

[0101] Accuracy Solution 3: Accurately measure 0.12 ml of irinotecan liposome injection into the prepared chromatography column, and prepare test solution A and test solution B according to "Example 1".

[0102] Inject the above accuracy solutions into the liquid chromatograph respectively and record the relevant results, see Table 7.

[0103] Table 7. Linear relationship test results

[0104]

[0105] The results showed that the recovery rate was between 98.0% and 102.0%, with good accuracy.

[0106] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for determining the encapsulation efficiency of irinotecan liposome injection, characterized in that: include: 1) separating the irinotecan liposome injection into an encapsulated body solution and a free body solution; 2) preparing a test solution A from the encapsulated body solution and preparing a test solution B from the free body solution, both of which include a heating step; 3) The irinotecan concentration C in the test solution A and the test solution B was tested by high performance liquid chromatography. A and C B , the encapsulation efficiency was calculated according to the following formula: Encapsulation efficiency (%) = C A / (C A +C B )*100%.

2. The method according to claim 1, wherein: The encapsulated bodies and free bodies were separated by gel column chromatography using agarose as the column material and 0.9% sodium chloride solution as the eluent.

3. The method according to claim 1, wherein: The preparation of test solution A and test solution B includes: 2-1) using 0.9% sodium chloride solution as an eluent, diluting the encapsulated body solution and the free body solution obtained in step 1) with a diluent to obtain diluents, respectively, wherein the diluent is one or more of methanol, isopropanol, acetonitrile and ethanol, wherein isopropanol is preferred; 2-2) ultrasonically treating the dilution for 3-10 minutes, preferably 4-5 minutes; 2-3) The diluted solution is formulated into a solution with a concentration of 0.04 to 12.9 μg / ml using a first solvent to obtain a test solution A and a test solution B, respectively, wherein the first solution is a mixed aqueous solution of sodium dihydrogen phosphate and 1-octane sodium sulfonate at pH 3.6, preferably, the concentration of sodium dihydrogen phosphate in the first solution is 2.8 g / L, and the concentration of sodium 1-octane sodium sulfonate is 1.8 g / L.

4. The method according to claim 3, wherein: When preparing the test solution A, the amount of the diluent used is 0.7-2 times, preferably 1-1.5 times, the volume of the encapsulation solution, and the volume ratio of the diluent to the first solvent is 1:3-4.

5. The method according to claim 3, wherein: When preparing test solution B, the amount of diluent used is 0.5-1 times the volume of the free body solution, and the volume ratio of the diluent to the first solvent is 1:2-3.

6. The method according to any one of claims 3 to 5, wherein: The heating temperature in step 2) is between 45-60° C. and the heating time is controlled between 30-50 minutes.

7. The method according to claim 3, wherein: The chromatographic column is an octadecylsilane bonded silica gel column, and the mobile phase is the first solution-acetonitrile-methanol, with a ratio of 40-60:10-20:15-25, preferably 53:20:

27.

8. The method according to claim 3, wherein: Column temperature: 35-45°C, preferably 40°C; Flow rate: 0.8-1.2 ml / min, preferably 1.0 ml / min; The detector is: an ultraviolet detector, with a detection wavelength of 235 to 255 nm, preferably 255 nm; Injection volume: 20μl.

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