CN119438468A - A method for determining the encapsulation efficiency of irinotecan liposome injection - Google Patents

Abstract

The present invention provides a method for determining the encapsulation rate of irinotecan liposome injection, which comprises: 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) respectively testing the irinotecan concentrations in the test solution A and the test solution B by high performance liquid chromatography. By 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.

Description

Method for measuring encapsulation efficiency of irinotecan liposome injection

Technical Field

The invention belongs to the field of synthesis of small molecular compounds, and particularly relates to a preparation method of an imidazole derivative.

Background

Irinotecan hydrochloride is a semisynthetic water-soluble camptothecin derivative, and is an antitumor drug marketed in 1994. Irinotecan hydrochloride has good water solubility, higher antitumor activity than camptothecin, and lower toxicity than camptothecin. At present, the traditional Chinese medicine composition is still one of main medicines for treating metastatic colon cancer and rectal cancer. The approved dosage forms are injections, and the main adverse reactions are neutropenia and diarrhea, which are difficult to be tolerated by patients after long-term large-dose application. The liposome injection can be prepared into liposome injection, so that adverse reaction can be improved, and the stability of the medicine can be improved.

The entrapment rate of a liposome refers to the percentage of the total amount of drug in the liposome suspension that is encapsulated by a substance (e.g., a drug). The liposome is an important index for controlling the quality of the liposome, reflects the encapsulation degree of the drug by a carrier, and has the percentage encapsulation rate of EN% = (1-C _f/C_t) multiplied by 100 percent, wherein Cf is the amount of free drug, and Ct is the total amount of the drug in liposome suspension. Therefore, the encapsulation efficiency is usually measured by separating the free drug from the liposome in the formulation, and then calculating the encapsulation efficiency. Liposomes are generally required to have drug encapsulation rates of over 80%.

There are several methods reported in the prior art for determining liposome encapsulation efficiency. Zhao Ligang et al (determination of encapsulation efficiency and study of drug efficacy of vinorelbine tartrate long-circulating liposome, university of medical science, 2010,41 (6): pp 508-512) determine encapsulation efficiency by ultrafiltration, but the use of the liposome is limited by possible aggregation of particles due to the action of centrifugal machine, and errors in measurement caused by possible adsorption of drugs by ultrafiltration membrane. The ultrafiltration method has high instrument requirement and high price. And high-speed centrifugation has poor separation effect on liposomes having a small particle size (< 100 nm), and the liposomes cannot be completely precipitated. Huang Junqin et al (preparation of gemcitabine hydrochloride liposome and determination of content and encapsulation efficiency, journal of antibiotics in China, 1 st year of 2010, 35 vol. 1 st, pp 30-32) adopts dialysis method to determine encapsulation efficiency, but the medicine packed in the liposome may leak out, and the method is not applicable to insoluble medicine and takes a long time. Zhang Jianjian et al (preparation of irinotecan hydrochloride liposome and in vitro drug release characteristic study, china pharmacy, volume 19, 10 th, pp762-764 of 2008) determine encapsulation rate by gel chromatography-ultraviolet spectrometry, but the ultraviolet measurement method has interference caused by auxiliary materials and impurities, and has inferior specificity to high performance liquid chromatography.

Zhang Xiaolei et al (research on compound irinotecan hydrochloride and fluorouracil liposome injection encapsulation efficiency determination method, china modern application pharmacy, volume 38, 13, 2021, 7, pp 1588-1593) determined encapsulation efficiency by gel chromatography, which uses gel column chromatography with phosphate buffer solution of pH 6.5 as eluent, under established HPLC-DAD chromatography conditions, kromasil C18 column (250 mm. Times.4.6 mm,5 μm) with methanol-pH 6.6 phosphate buffer (5:95) as mobile phase A, and methanol-pH 6.6 phosphate buffer (60:40) as mobile phase B, gradient elution. The column temperature is 35 ℃ and the flow rate is 1.0mL min < -1 >. The detection wavelengths were 255,268nm, respectively. CN116718696a discloses a method for determining encapsulation efficiency of liposome injection, which adopts Sepharose CL-4B agarose gel chromatography to separate encapsulated irinotecan and free irinotecan. According to this method and according to the actual conditions, irinotecan Kang Feng in the test solution B for measuring the encapsulation efficiency is unstable in area, probably due to the intramolecular lactone conversion process to carboxylic acid type structure of irinotecan. Both methods employ high performance liquid chromatography, but the applicant has found that stable test results cannot be obtained under such measurement conditions, and that the measured results vary with the time of the test solution being left for a while under other conditions. This may be related to the instability of irinotecan under test conditions.

Disclosure of Invention

The invention aims to solve the problem that the measurement result is unstable when the encapsulation efficiency of irinotecan liposome injection is analyzed by adopting a liquid chromatography analysis method in the prior art.

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

1) Separating irinotecan liposome injection into an encapsulation body solution and a free body solution;

2) Preparing a test solution A from the encapsulating body solution, and preparing a test solution B from the free body solution, wherein the test solution A and the free body solution all comprise a heating step;

3) Irinotecan concentrations C _A and C _B in test solution a and test solution B, respectively, were tested by high performance liquid chromatography, encapsulation efficiency was calculated according to the following formula,

Encapsulation efficiency (%) =c _A/(C_A+C_B) ×100%.

In one embodiment of the invention, the encapsulates and the free bodies are separated by gel column chromatography using agarose as the column material and 0.9% sodium chloride solution as the eluent.

In one embodiment of the present invention, the preparation of test solution A and test solution B comprises:

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

2-2) sonicating the dilution for 3-10 minutes, preferably 4-5 minutes;

2-3) preparing the diluent into a solution with the concentration of 0.04-12.9 mug/ml by using a first solvent to respectively obtain a test solution A and a test solution B, wherein the first solution is a mixed aqueous solution of sodium dihydrogen phosphate with the pH of 3.6 and sodium 1-octyl sulfonate, and preferably, the concentration of sodium dihydrogen phosphate in the first solution is 2.8g/L and the concentration of sodium 1-octyl sulfonate is 1.8g/L.

Preferably, the test solution A is prepared by diluting the first solvent with a diluent in an amount of 0.7 to 2 times, preferably 1 to 1.5 times, the volume ratio of the diluent to the first solvent being 1.3 to 4.

Preferably, the diluent is used in an amount of 0.5 to 1 times the volume of the free solution, and the volume ratio of the diluent to the first solvent is 1.2 to 3.

Preferably, the heating temperature in step 2) is between 45 and 60 ℃ and the time is controlled between 30 and 50 minutes.

In one embodiment of the invention, the following analytical conditions are used:

the chromatographic column is octadecylsilane chemically bonded silica gel column;

the mobile phase is a first solution, acetonitrile-methanol, in a ratio of 40-60:10-20:15-25, wherein the preferable ratio is 53:20:27;

the column temperature is 35-45 ℃, preferably 40 ℃;

The flow rate is 0.8-1.2 ml/min, preferably 1.0ml/min;

The detector is an ultraviolet detector, and the detection wavelength is 235-255 nm, preferably 255nm;

The sample injection amount is 20 μl.

By adopting the method, the detection of the encapsulation efficiency of the irinotecan liposome injection can be realized only by high performance liquid chromatography, and the problem of unstable test solution is solved.

Drawings

Some specific embodiments of the invention will be described in detail hereinafter by way of example and not by way of limitation with reference to the accompanying drawings. The same reference numbers will be used throughout the drawings to refer to the same or like parts or portions. It will be appreciated by those skilled in the art that the drawings are not necessarily drawn to scale. In the accompanying drawings:

FIG. 1 is a detection chromatogram of example 1 for 5 test solutions B at different time intervals;

FIG. 2 is a detection chromatogram of comparative example 1 for test solution B at 5 different holding times.

Detailed Description

In order to make the objects, features and advantages of the present invention more comprehensible, the technical solutions of the present invention will be clearly described with reference to the following specific examples and accompanying drawings, and it is obvious that the described examples are illustrative only and not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

One of the features of the present invention is that the sample solution is subjected to a heat treatment before being subjected to high-phase liquid chromatography. The inventors found that the sample solution after the heat treatment was stable under the test conditions and the concentration change was not detected with the change in the standing time.

The method according to the invention comprises the following steps:

1) Separating irinotecan liposome injection into an encapsulation body solution and a free body solution;

2) Preparing a test solution A from the encapsulating body solution, and preparing a test solution B from the free body solution, wherein the test solution A and the free body solution all comprise a heating step;

3) Irinotecan concentrations C _A and C _B in test solution a and test solution B, respectively, were tested, encapsulation efficiency was calculated according to the following formula,

Encapsulation efficiency (%) =c _A/(C_A+C_B) ×100%.

Preparation of irinotecan liposome injection

In the present invention, irinotecan liposomes can be prepared by methods 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, with an ammonium sulfate gradient method being preferred. In the method, the prescription amount of cholesterol and the pezited phosphatidylethanolamine are added with proper amount of ethanol for ultrasonic dissolution, and the ethanol is removed by reduced pressure evaporation. An appropriate amount of ammonium sulfate aqueous solution was added and the film was washed with a rotary evaporator to completely hydrate. The hydrated mixture is circularly treated by an ultrafiltration system, and the aqueous phase ammonium sulfate outside the liposome is removed and replaced by an aqueous solution. Adding the raw material liquid into a water bath kettle for heating and incubating, and then filtering by a microporous filter membrane.

In a typical embodiment of the present invention, irinotecan hydrochloride liposome injection is used in the homemade of applicant's company.

Separation of the encapsulation from the episome

In the present invention, the encapsulated irinotecan (i.e., the encapsulate) is first separated from the unencapsulated portion (the free form) in the formulation, and the preferred separation method is gel column chromatography. In order to prepare the chromatographic column, proper amount of gel can be taken, soaked and washed by deionized water, the upper layer aqueous solution is discarded, and the gel is slowly filled into a chromatographic column tube to a certain height and then is balanced by an eluent.

The chromatographic column material used for separation can be agarose or a derivative thereof, or one of sephadex. In one embodiment, the encapsulates and the free bodies are separated using an agarose gel column with 0.9% sodium chloride solution as eluent. During elution, the encapsulated body solution is obtained first, and then the elution is continued to obtain the free body solution.

In another embodiment, the column is prepared with a G-50 sephadex.

Preparation of test solution A

Next, the obtained encapsulated solution was prepared as a test solution a, and the solvent in the encapsulated solution was the eluent used in the previous step, i.e., 0.9% sodium chloride solution, in an amount of elution. In a typical embodiment, to isolate the liposome injection, an encapsulate solution may be obtained by eluting with 50-100 volumes of eluent. The solution is suitably diluted with a diluent which is one or more of methanol, isopropanol, acetonitrile and ethanol, with isopropanol being preferred. Isopropanol can disrupt the structure of the liposome, freeing the encapsulated irinotecan to be detected by the detector. The diluent may be used in an amount of 0.7 to 2 times, preferably 1 to 1.5 times the volume of the encapsulate solution. The diluent is then preferably sonicated for 3 to 10 minutes, more preferably 4 to 5 minutes, with the purpose of further promoting demulsification.

Next, the solution is formulated with the first solvent into a predetermined concentration range such that irinotecan in test solution a falls within a detectable linear range. In the invention, the concentration of irinotecan in the test solution A is suitably in the range of 0.04-12.9 mug/ml. The first solution was an aqueous solution comprising sodium dihydrogen phosphate and sodium 1-octanesulfonate at a pH of 3.6. In typical embodiments, the concentration of sodium dihydrogen phosphate in the first solution is 2.0 to 4.0g/L, preferably 2.8g/L, and the concentration of sodium 1-octanesulfonate is 1.0 to 3.0g/L, preferably 1.8g/L. The volume ratio of the diluent to the first solvent may be in the range of 1..3 to 4.

The test solution A prepared by the method is not only suitable for testing under the high performance liquid chromatography condition, but also can be well stabilized after being subjected to heat treatment. In an exemplary embodiment of the invention, water bath heating is employed, with the water bath temperature set between 45-60 ℃, preferably between 50-55 ℃. The time is controlled between 30 and 50 minutes, preferably between 30 and 40 minutes.

Preparation of test solution B

Next, the obtained free solution was prepared as a test solution B, in which the solvent was 0.9% sodium chloride solution in an amount of elution. In a typical embodiment, the liposome injection is isolated and eluted with 50-100 volumes of eluent to obtain a free solution. The solution is suitably diluted with a diluent which is one or more of methanol, isopropanol, acetonitrile and ethanol, with isopropanol being preferred. The diluent may be used in an amount of 0.5 to 1 times the volume of the free solution. Then, the solution is formulated with the above-mentioned first solvent into a predetermined concentration range such that irinotecan in the test solution B falls within a detectable linear range. The volume ratio of the diluent to the first solvent may be in the range of 1..2 to 3.

The test solution B prepared by the method is not only suitable for testing under the high performance liquid chromatography condition, but also can be well stabilized after being subjected to heat treatment. In an exemplary embodiment of the present invention, water bath heating is employed, the water bath temperature is set between 45-60 ℃ and the time is controlled between 30-50 minutes.

Preparation of control solution

Taking irinotecan hydrochloride reference substance, dissolving the irinotecan hydrochloride reference substance into 0.4-0.5mg/ml solution by using methanol to obtain irinotecan stock solution, and freezing and storing the stock solution, and further diluting the irinotecan stock solution into 0.04-12.9 mug/ml reference substance solution by using methanol when in use.

Liquid chromatography detection

The chromatographic conditions for detecting the test solutions A and B are as follows:

Chromatographic column with octadecylsilane chemically bonded silica gel as filler;

the ratio of the mobile phase to the first solution-acetonitrile-methanol is 40-60:10-20:15-25, wherein the preferable ratio is 53:20:27

The column temperature is 35-45 ℃, preferably 40 ℃;

The flow rate is 0.8-1.2 ml/min, preferably 1.0ml/min;

The detector is an ultraviolet detector, and the detection wavelength is 235-255 nm, preferably 255nm;

The sample injection amount is 20 μl.

Example 1

Preparing a separation column, namely taking 20ml of agarose gel CL-4B into a measuring cylinder, soaking and stirring the agarose gel CL-4B in deionized water, standing and layering the agarose gel CL-4B, discarding an upper aqueous solution, repeatedly cleaning the agarose gel CL-4B for 4 to 5 times, and slowly pouring the cleaned agarose gel CL-4B aqueous solution into the chromatography column, wherein the height of a column bed is about 15cm. Washing with deionized water, standing for 10 min, opening liquid outlet at lower end of chromatographic column, draining liquid in the column, taking out excessive agarose gel CL-4B to make the height of column bed be 12cm, and washing agarose gel CL-4B with 0.9% sodium chloride solution for balancing.

The preparation of the test solution A comprises precisely measuring 0.1ml of irinotecan liposome injection into a prepared chromatographic column, adding 0.25ml of 0.9% sodium chloride solution after the sample completely enters the column bed, adding 7.75ml of 0.9% sodium chloride solution after the sample completely enters the column bed, taking the solution by a 50ml measuring flask, adding 10ml of isopropanol, carrying out ultrasonic treatment for 5min, diluting to a scale by the first solution, placing in a 50 ℃ water bath, heating for 30min, cooling to room temperature, and shaking uniformly to obtain the test solution A.

B, adding 7ml of 0.9% sodium chloride solution after the step B is completed, taking the solution by using a 20ml measuring flask, adding 4ml of isopropanol, carrying out ultrasonic treatment for 5min, diluting to a scale by using the first solution, heating in a water bath at 50 ℃ for 30min, taking out, cooling to room temperature, and shaking uniformly to obtain the test solution B.

The preparation of the reference substance solution comprises the steps of precisely weighing 43mg of irinotecan hydrochloride reference substance, placing in a 100ml measuring flask, adding a proper amount of methanol for dissolution, and then fixing the volume to a scale mark to obtain the irinotecan stock solution, and preparing the irinotecan stock solution into the reference substance solution with the methanol of 0.04-12.9 mug/ml.

The chromatographic analysis is to detect the test solutions A and B by adopting a high performance liquid chromatography, wherein the chromatographic conditions are chromatographic columns (4.6 mm multiplied by 250mm,5 mu m) adopting octadecylsilane bonded silica gel as a filler, solution 1 (mixed aqueous solution of sodium dihydrogen phosphate and sodium 1-octanesulfonate) -acetonitrile-methanol (53:20:27) is adopted as a mobile phase, the flow rate is 1.0ml per minute, the column temperature is 40 ℃, the detection wavelength is 255nm, and the sample injection volume is 20 mu l.

The peak area of the test solution B after being left at room temperature for various times was measured, and the test results are shown in Table 1 and FIG. 1.

Table 1. Test solution B was tested for peak area at different times of placement:

As can be seen from Table 1 and FIG. 1, the test solutions prepared by the method of the present invention were stable under system conditions and at room temperature and did not undergo chemical changes.

Six parallel tests were performed with test solutions A and B, respectively, and the test results are shown in Table 2:

TABLE 2 parallel test results of EXAMPLE 1

Example 2

The procedure of example 1 was repeated except that the gel in step a was G-50 dextran gel, the bed height was 11.5cm, and the rest was the same as in example 1. Two parallel experiments were performed and the results are shown in Table 3.

TABLE 3 test results of example 2

Comparative example 1:

Step c of example 1 was repeated to prepare a test solution B, except that the heating step was omitted. The peak area of solution B after standing at room temperature for various times was measured and the test results are shown in table 4 and fig. 2.

TABLE 4 Peak area test results of comparative example 1 test solution B

As can be seen from Table 4 and FIG. 2, the test solutions not prepared by the method of the present invention are unstable under storage conditions.

EXAMPLE 3 methodological verification

1. Sample injection precision test

Preparing reference stock solution, taking appropriate amount of irinotecan hydrochloride reference substance, precisely weighing, adding methanol for dissolving and diluting to obtain solution containing 0.43mg of irinotecan per 1 ml. And diluting the reference substance stock solution to 0.04-12.9 mug/ml of reference substance solution by using methanol. The test was performed in 6 parallel runs following the liquid chromatography conditions in example 1. The test results are shown in Table 5.

TABLE 5 sample injection precision test results

The result shows that 6 needle reference substance solutions are continuously injected under the method, the peak area RSD is 0.1%, the retention time RSD is 0%, and the injection precision is good.

2. Test of Linear relation

The reference stock solution is prepared by taking a proper amount of irinotecan hydrochloride reference, precisely weighing, adding methanol for dissolving and diluting to prepare a solution containing 0.43mg of irinotecan in each 1 ml.

And (2) diluting the reference substance stock solution to 0.04-12.9 mug/ml of reference substance solution by using methanol. The liquid chromatography conditions in example 1 were used to test, record the chromatograms, and the linear regression calculation was performed with the peak area logarithmic value as the ordinate and the concentration logarithmic value as the abscissa, and the results are shown in Table 6.

TABLE 6 test results of linear relationship

The result shows that the concentration and the peak area have good linear relation in the range of 0.0443-13.3044 mug/ml, the linear equation y=67332.1100x+249.8059, and the correlation coefficient r is 1.000>0.999.

3. Accuracy of

Preparation of accuracy solution:

Accuracy solution 1. Precision measurement of irinotecan liposome injection 0.08ml into prepared chromatographic column, test solution A and test solution B were prepared as in "example 1".

Accuracy solution 2. Precision measurement of irinotecan liposome injection 0.1ml into prepared chromatographic column, test solution A and test solution B were prepared as in "example 1".

Accuracy solution 3. Precision measurement of irinotecan liposome injection 0.12ml into prepared chromatographic column, test solution A and test solution B were prepared as in "example 1".

The above accuracy solutions were injected into the liquid chromatograph, respectively, and the results were recorded as shown in table 7.

TABLE 7 test results of linear relationship

The result shows that the recovery rate is 98.0-102.0%, and the accuracy is good.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (8)

1. The method for measuring the encapsulation efficiency of the irinotecan liposome injection is characterized by comprising the following steps of:

1) Separating irinotecan liposome injection into an encapsulation body solution and a free body solution;

2) Preparing a test solution A from the encapsulating body solution, and preparing a test solution B from the free body solution, wherein the test solution A and the free body solution all comprise a heating step;

3) Irinotecan concentrations C _A and C _B in test solution a and test solution B, respectively, were tested by high performance liquid chromatography, 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 encapsulate and the free body are separated by gel column chromatography using agarose as column material and 0.9% sodium chloride solution as eluent.

3. The method of claim 1, wherein the preparation of test solution a and test solution B comprises:

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

2-2) sonicating the dilution for 3-10 minutes, preferably 4-5 minutes;

2-3) preparing the diluent into a solution with the concentration of 0.04-12.9 mug/ml by using a first solvent to respectively obtain a test solution A and a test solution B, wherein the first solution is a mixed aqueous solution of sodium dihydrogen phosphate with the pH of 3.6 and sodium 1-octyl sulfonate, and preferably, the concentration of sodium dihydrogen phosphate in the first solution is 2.8g/L and the concentration of sodium 1-octyl sulfonate is 1.8g/L.

4. A method according to claim 3, wherein the test solution a is prepared with a diluent in an amount of 0.7 to 2 times, preferably 1 to 1.5 times the volume of the solution of the encapsulating body, and the diluent and the first solvent are diluted in a volume ratio of 1..3 to 4.

5. The method according to claim 3, wherein the diluent is used in an amount of 0.5 to 1 times the volume of the free solution, and the volume ratio of the diluent to the first solvent is 1.2 to 3.

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

7. A method according to claim 3, wherein the chromatographic column is an octadecylsilane chemically bonded silica column and the mobile phase is a first solution-acetonitrile-methanol in a ratio of 40-60:10-20:15-25, preferably 53:20:27.

8. The method of claim 3, wherein,

The column temperature is 35-45 ℃, preferably 40 ℃;

The flow rate is 0.8-1.2 ml/min, preferably 1.0ml/min;

The detector is an ultraviolet detector, and the detection wavelength is 235-255 nm, preferably 255nm;

The sample injection amount is 20 μl.