A method for determining the encapsulation efficiency of liposome injection solutions

Abstract

This invention relates to a method for determining the encapsulation efficiency of liposome injection solutions. The method comprises the following steps: 1) preparation of a blank solution; 2) preparation of a test solution; 3) determination: injecting the blank solution and the test solution into a high-performance liquid chromatograph (HPLC) to obtain a chromatogram, and calculating the encapsulation efficiency of the test solution based on the peak area of ​​the chromatogram. The chromatographic conditions for the HPLC are as follows: instrument: HPLC system, VWD or DAD; column: Thermo Hypersil BDS C8; mobile phase: sodium hexanesulfonate solution: acetonitrile = 75:25; column temperature: 40℃; flow rate: 1.0 ml / min; detection wavelength: 255 nm; injection volume: 10 μl; run time: 10 min.

Description

Technical Field

[0001] This invention relates to a method for detecting the active ingredient in a drug, specifically a method for determining the encapsulation efficiency of liposomes. Background Technology

[0002] Encapsulation efficiency refers to the percentage of encapsulated substance (such as a drug) in a liposome suspension relative to the total drug amount. It is an important indicator for liposome quality control, reflecting the degree to which the drug is encapsulated by the carrier. Encapsulation efficiency = (Drug encapsulated in liposomes / Total drug amount in liposomes) × 100%. The percentage encapsulation efficiency can be calculated using the following formula: EN% = (1 - Cf / Ct) × 100%, where Cf is the amount of free drug and Ct is the total drug amount in the liposome suspension.

[0003] Generally, the encapsulation efficiency can be analyzed after separating the free drug from the liposomes. Separation methods such as dextran gel electrophoresis, ultracentrifugation, and dialysis can be used to separate the free drug and liposomes in solution, and the encapsulation efficiency can be calculated separately. Typically, a drug encapsulation efficiency of over 80% is required for the liposomes. Irinotecan hydrochloride liposome injection, a marketed drug, is a topoisomerase 1 inhibitor encapsulated in lipid bilayer vesicles or liposomes. It is indicated for metastatic pancreatic cancer and is used in combination with 5-fluorouracil / leucovorin to treat patients with advanced (metastatic) pancreatic cancer who have previously received gemcitabine chemotherapy.

[0004] This invention provides a new method for determining the encapsulation efficiency of liposomes, which is particularly suitable for irinotecan hydrochloride liposome injection. Summary of the Invention

[0005] This invention provides a method for determining the encapsulation efficiency of liposome injection solutions, characterized in that the method comprises the following steps:

[0006] 1) Preparation of blank solution;

[0007] 2) Preparation of test solutions; including preparation of test solution A; preparation of test solution B; preparation of test solution C;

[0008] 3) Determination: Take blank solution and test solution, inject them into high performance liquid chromatograph, obtain chromatograms, and calculate the encapsulation rate of test solution based on the peak area of ​​chromatogram;

[0009] The calculation method uses the following formula:

[0010] (1) Encapsulation ratio:

[0011]

[0012] (2) Column recovery rate:

[0013]

[0014] In the formula: A1 represents the peak area of ​​the drug in the chromatogram of test solution A; A2 represents the peak area of ​​the drug in the chromatogram of test solution B; A3 represents the peak area of ​​the drug in the chromatogram of test solution C.

[0015] The chromatographic conditions for the high-performance liquid chromatography are as follows:

[0016] Instrument: Liquid Chromatograph, VWD or DAD

[0017] Column: Thermo Hypersil BDS C8 (4.6mm × 150mm, 5μm)

[0018] Mobile phase: Sodium 1-hexanesulfonate solution: Acetonitrile = 75:25

[0019] Column temperature: 40℃; flow rate: 1.0 ml / min; detection wavelength: 255 nm;

[0020] Injection volume: 10 μl; Run time: 10 min.

[0021] The test sample is irinotecan liposome injection.

[0022] The test sample may also be other liposome injection solutions, such as doxorubicin hydrochloride liposome injection, amphotericin B liposome injection, paclitaxel liposome injection, bupivacaine liposome injection, etc., as needed.

[0023] The blank solution is prepared as follows:

[0024] Preparation of blank solution 1: Take the diluent and label it blank solution 1.

[0025] Preparation of Blank Solution 2: Accurately measure 100 μl of eluent and place it in the chromatography column. After it has completely entered the column bed, immediately add 0.5 ml of eluent. Once this portion of eluent has also entered the column bed, add 4.5 ml of eluent and elute under gravity. Collect the eluent in a 50 ml volumetric flask, add another 10 ml of eluent, and finally dilute to the mark with diluent. Shake well and label as Blank Solution 2.

[0026] Preparation of blank solution 3: Accurately add 15 ml of eluent to the chromatography column after the previous elution, and elute under gravity. Collect the eluent in a 50 ml volumetric flask, dilute to the mark with diluent, and mix well. Label this as blank solution 3.

[0027] The chromatography column is a gel chromatography column, which is prepared as follows: take an appropriate amount of Sepharose CL-4B agarose gel, remove air bubbles by sonication, and then slowly pack it into the chromatography column tube to a height of about 8 cm. Then, equilibrate it with about 20 ml of eluent for later use.

[0028] The preparation method for the test solution is as follows:

[0029] Preparation of test solution A: Accurately measure 100 μl of the test sample and place it in a chromatography column. Once the sample has completely entered the column bed, immediately add 0.5 ml of eluent. After this portion of eluent has also entered the column bed, add 4.5 ml of eluent and elute under gravity. Collect the eluent in a 50 ml volumetric flask, add another 10 ml of eluent, and finally dilute to the mark with diluent. Shake well and filter through a 0.22 μm filter membrane. Take an appropriate amount of the filtrate; this is test solution A.

[0030] Preparation of test solution B: Accurately add 15 ml of eluent to the chromatography column after the previous elution, and elute under gravity. Collect the eluent in a 50 ml volumetric flask, dilute to the mark with diluent, mix well, and filter through a 0.22 μm filter membrane. Take an appropriate amount of the filtrate as test solution B. Preparation of test solution C: Accurately measure 100 μl of the test sample, place it in a 50 ml volumetric flask, add 15 ml of eluent, dilute to the mark with diluent, mix well, filter through a 0.22 μm filter membrane, and take an appropriate amount of the filtrate, labeling it as test solution C.

[0031] The eluent is prepared as follows: Weigh 9g of sodium chloride, add an appropriate amount of water to dissolve and dilute to 1000ml, shake well. The diluent is prepared as follows: Measure 1ml of hydrochloric acid, add methanol to dilute to 1000ml, and shake well.

[0032] The determination method is as follows: Take 10 μL each of blank solutions 1, 2, and 3 and test solutions A, B, and C, inject them into a high-performance liquid chromatograph, obtain chromatograms, and calculate the encapsulation rate of the test solutions based on the peak areas of the chromatograms;

[0033] The calculation method uses the following formula:

[0034] (1) Encapsulation ratio:

[0035]

[0036] (2) Column recovery rate:

[0037]

[0038] In the formula: A1 represents the peak area of ​​irinotecan in the chromatogram of test solution A; A2 represents the peak area of ​​irinotecan in the chromatogram of test solution B; A3 represents the peak area of ​​irinotecan in the chromatogram of test solution C.

[0039] The most preferred method of the present invention comprises the following steps:

[0040] 1) Preparation of blank solution:

[0041] Preparation of blank solution 1: Take the diluent and label it as blank solution 1.

[0042] Preparation of blank solution 2: Accurately measure 100 μl of eluent and place it in the chromatography column. After it has completely entered the column bed, immediately add 0.5 ml of eluent. After this part of the eluent has also entered the column bed, add 4.5 ml of eluent and elute under gravity. Collect the eluent in a 50 ml volumetric flask, add another 10 ml of eluent, and finally dilute to the mark with diluent. Shake well and label it as blank solution 2.

[0043] Preparation of blank solution 3: Accurately add 15 ml of eluent to the chromatography column after the previous elution, and elute under gravity. Collect the eluent in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, and label it as blank solution 3.

[0044] The chromatography column is a gel chromatography column, which is prepared as follows: take an appropriate amount of Sepharose CL-4B agarose gel, remove air bubbles by sonication, and then slowly pack it into the chromatography column tube to a height of about 8 cm. Then, equilibrate it with about 20 ml of eluent for later use.

[0045] 2) Preparation of the test solution:

[0046] Preparation of test solution A: Accurately measure 100 μl of the test sample and place it in the chromatography column. After it has completely entered the column bed, immediately add 0.5 ml of eluent. After this portion of eluent has also entered the column bed, add 4.5 ml of eluent and elute under gravity. Collect the eluent in a 50 ml volumetric flask, add another 10 ml of eluent, and finally dilute to the mark with diluent. Shake well and filter through a 0.22 μm filter membrane. Take an appropriate amount of the filtrate as test solution A.

[0047] To prepare test solution B, precisely add 15 ml of eluent to the chromatography column after the previous elution, and elute under gravity. Collect the eluent in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, filter through a 0.22 μm filter membrane, and take an appropriate amount of the filtrate as test solution B.

[0048] Preparation of test solution C: Accurately measure 100 μl of the test sample and place it in a 50 ml volumetric flask. Add 15 ml of eluent and dilute to the mark with diluent. Shake well and filter through a 0.22 μm filter membrane. Take an appropriate amount of the filtrate and label it as test solution C.

[0049] The eluent is prepared as follows: Weigh 9g of sodium chloride, add an appropriate amount of water to dissolve it, and dilute to 1000ml. Shake well. The diluent is prepared as follows: Measure 1ml of hydrochloric acid, add methanol to dilute to 1000ml, and shake well.

[0050] 3) Determination: Take 10 μL each of blank solutions 1, 2, and 3 and test solutions A, B, and C, inject them into the high performance liquid chromatograph, obtain the chromatogram, and calculate the encapsulation rate of the test solution based on the peak area of ​​the chromatogram;

[0051] The calculation method uses the following formula:

[0052] (1) Encapsulation ratio:

[0053]

[0054] (2) Column recovery rate:

[0055]

[0056] In the formula: A1 represents the peak area of ​​irinotecan in the chromatogram of test solution A; A2 represents the peak area of ​​irinotecan in the chromatogram of test solution B; A3 represents the peak area of ​​irinotecan in the chromatogram of test solution C.

[0057] The chromatographic conditions for the high-performance liquid chromatography are as follows:

[0058] Instrument: Liquid Chromatograph, VWD or DAD

[0059] Column: Thermo Hypersil BDS C8 (4.6mm × 150mm, 5μm)

[0060] Mobile phase: Sodium 1-hexanesulfonate solution (weigh 2g of sodium 1-hexanesulfonate, dissolve in 1000ml of water, add 2ml of triethylamine, mix well, and adjust the pH to 2.5 with phosphoric acid): Acetonitrile = 75:25

[0061] Column temperature: 40℃; flow rate: 1.0 ml / min; detection wavelength: 255 nm;

[0062] Injection volume: 10 μl; Run time: 10 min.

[0063] The test sample is irinotecan liposome injection.

[0064] The chromatographic conditions (selection of mobile phase ratio) of this invention were obtained through screening, and the screening process is as follows:

[0065] The effects of different mobile phase ratios (10% acetonitrile, 25% acetonitrile, and 50% acetonitrile) on chromatographic peaks were investigated. The results showed that 10% acetonitrile resulted in no peaks, while 50% acetonitrile eluted too early, failing to achieve complete separation from the solvent method, and exhibiting poor peak shape. 25% acetonitrile showed excellent peak shape and good separation from the solvent peak; therefore, 15% acetonitrile was the preferred choice.

[0066] The difference between this detection method and existing similar methods, such as those in application number CN201110263566, is that they combine high-performance liquid chromatography (HPLC) with size exclusion chromatography (SUC) to determine the encapsulation efficiency of liposome drugs. This method is particularly suitable for nanoliposome formulations encapsulating hydrophobic and lipophobic drugs. However, its preparation and detection processes are complex, requiring the connection of a size-exclusion silica column to the HPLC phase, and necessitating the replacement of the mobile phase during testing. This invention uses gel permeation chromatography (GPC) to achieve a simple and complete separation of the encapsulated and free components. Furthermore, the HPLC method eliminates the need for mid-process mobile phase changes, making it highly efficient and convenient.

[0067] The advantages of this invention are:

[0068] 1. The sample extraction method of this invention is simple and efficient;

[0069] 2. This invention has a short detection time and high inspection efficiency. Attached Figure Description

[0070] Figure 1 Spectrum of test solution A

[0071] Figure 2 Spectrum of test solution B

[0072] Figure 3 C-chromatogram of the test solution Detailed Implementation

[0073] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.

[0074] Example 1

[0075] The test sample is irinotecan liposome injection.

[0076] 1) Chromatographic conditions:

[0077] Instrument: Liquid Chromatograph, VWD or DAD

[0078] Column: Thermo Hypersil BDS C8 (4.6mm × 150mm, 5μm)

[0079] Mobile phase: Sodium 1-hexanesulfonate solution (weigh 2g of sodium 1-hexanesulfonate, dissolve in 1000ml of water, add 2ml of triethylamine, mix well, and adjust the pH to 2.5 with phosphoric acid): Acetonitrile = 75:25

[0080] Column temperature: 40℃; flow rate: 1.0 ml / min; detection wavelength: 255 nm;

[0081] Injection volume: 10 μl; Run time: 10 min.

[0082] 2) Solution preparation:

[0083]

[0084]

[0085] 3) Determination: Take 10 μL of blank solution and 10 μL of test solution and inject them into the high performance liquid chromatograph to obtain the chromatogram. Calculate the encapsulation efficiency of the test solution based on the peak area of ​​the chromatogram.

[0086] 4) Test results:

[0087]

[0088]

Claims

1. A method for determining the encapsulation efficiency of irinotecan liposome injection, characterized by, The method, steps as follows: 1) preparation of blank solution: Preparation of blank solution 1: take diluent, labeled as blank solution 1, Preparation of blank solution 2: accurately take eluent 100 μl, place the chromatographic column, wait for complete into the bed, immediately supplement eluent 0.5 ml, when the part of eluent also into the bed, add eluent 4.5 ml, make it elute under the action of gravity, collect the eluent in 50 ml volumetric flask, add eluent 10 ml, finally dilute to the mark with diluent, shake, labeled as blank solution 2, Preparation of blank solution 3: in the completion of the last elution of chromatographic column, accurately add eluent 15 ml, make it elute under the action of gravity, collect the eluent in 50 ml volumetric flask, dilute to the mark with diluent, shake, labeled as blank solution 3, Wherein the chromatographic column is gel chromatography column, its preparation method is: take Sepharose CL-4B agarose gel, ultrasonic remove air bubble, then slowly fill in chromatographic column tube, fill gel height 8 cm, then balance with eluent 20 ml, standby, 2) preparation of test solution: Preparation of test solution A: accurately take test product 100 μl, place the chromatographic column, wait for complete into the bed, immediately supplement eluent 0.5 ml, when the part of eluent also into the bed, add eluent 4.5 ml, make it elute under the action of gravity, collect the eluent in 50 ml volumetric flask, add eluent 10 ml, finally dilute to the mark with diluent, shake, filter with 0.22 μm filter membrane, take the filtrate for test solution A, Preparation of test solution B: in the completion of the last elution of chromatographic column, accurately add eluent 15 ml, make it elute under the action of gravity, collect the eluent in 50 ml volumetric flask, dilute to the mark with diluent, shake, filter with 0.22 μm filter membrane, take the filtrate for test solution B, Preparation of test solution C: accurately take test product 100 μl, place in 50 ml volumetric flask, add eluent 15 ml, dilute to the mark with diluent, shake, filter with 0.22 μm filter membrane, take the filtrate for test solution C, Wherein, the preparation method of eluent is: take sodium chloride 9 g, add water to dissolve and dilute to 1000 ml, shake, the preparation method of diluent is: take hydrochloric acid 1 ml, dilute to 1000 ml with methanol, shake, 3) determination: take blank solution 1, 2, 3 and test solution A, B, C 10 ul, inject into high performance liquid chromatograph, get chromatogram, according to the peak area of chromatogram, calculate the encapsulation efficiency of test solution; The calculation method uses the following formula: (1) encapsulation efficiency: (2) chromatographic column recovery rate: In the formula: A1 represents the peak area of irinotecan in test solution A chromatogram; A2 represents the peak area of irinotecan in test solution B chromatogram; A3 represents the peak area of irinotecan in test solution C chromatogram, Wherein, the chromatographic conditions of high performance liquid chromatography are as follows: Instrument: liquid chromatograph, VWD or DAD Column: Thermo Hypersil BDS C8, 4.6mm x 150mm, 5μm, mobile phase: 1-hexanesulfonic acid sodium solution: acetonitrile = 75:25 Column temperature: 40℃; flow rate: 1.0ml / min; detection wavelength 255nm; Injection volume: 10μl; running time: 10min, The preparation method of 1-hexanesulfonic acid sodium solution is as follows: 1-hexanesulfonic acid sodium 2g is weighed, 1000ml of water is added to dissolve, 2ml of triethylamine is added, and the pH value is adjusted to 2.5 with phosphoric acid.

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