Method for determining related substances of lipidosome injection

Abstract

The invention relates to a method for determining irinotecan hydrochloride liposome injection related substances, which comprises the following steps: step 1, preparing a system applicability solution; step 2, preparing a sensitivity solution; step 3, preparing a test solution; and step 4, determination: taking the system applicability solution, the sensitivity solution and the test solution, injecting into a high performance liquid chromatograph to obtain a chromatogram, and calculating the content of impurities in the test solution according to the peak area of the chromatogram and an area normalization method.

Description

Technical Field

[0001] The invention relates to a drug detection method, in particular to a method for determining related substances of irinotecan hydrochloride liposome injection. Background Art

[0002] The chemical formula of irinotecan hydrochloride is C 33 H 38 N 4 O 6 ·HCl·3H 2 O, the structural formula is as follows:

[0003] Irinotecan is an off-white or light yellow powder. It is a derivative of natural camptothecin with a modified chemical structure. It can be used to prepare injections and is mainly used to treat digestive system tumors such as gastric cancer, colon cancer, rectal cancer, lung cancer, etc. It is also used to treat leukemia, hydatidiform mole and choriocarcinoma.

[0004] Irinotecan hydrochloride liposome injection is a high-end liposome preparation, with 43 mg of irinotecan per 10 ml. Irinotecan has different degrees of effect on lung cancer (small cell lung cancer, non-small cell lung cancer), colon cancer, rectal cancer, ovarian cancer, cervical cancer, gastric cancer, lymphoma, breast cancer, leukemia, etc., especially on small cell lung cancer and colon cancer, which is more active than the current anticancer drugs.

[0005] The main impurities of irinotecan are camptothecin, impurities A, B, and E.

[0006] Among them, the structure of impurity E is as follows:

[0007]

[0008] Since irinotecan and impurity E have the most similar structures, they are the most difficult to separate.

[0009] The following Chinese patents all disclose, to varying degrees, methods for determining isomeric impurities in irinotecan hydrochloride liposome injection.

[0010]

[0011] However, there is no report on the method for determining the related substances of irinotecan hydrochloride liposome injection in the prior art.

[0012] In view of the deficiencies of the prior art, the present invention screens and obtains a high performance liquid chromatography method for determining related substances in a high-end preparation of irinotecan hydrochloride liposome injection. It is unexpectedly found that the method of the present invention is superior to the prior art in various technical indicators. Summary of the invention

[0013] The present invention provides a method for determining related substances in irinotecan hydrochloride liposome injection, wherein the method comprises the following steps:

[0014] Step 1, preparation of system suitability solution: taking irinotecan hydrochloride reference substance and impurity E reference substance, adding methanol to prepare solutions containing 100-300 μg irinotecan hydrochloride and 0.1-0.5 μg impurity E per 1 ml;

[0015] Step 2, preparation of sensitivity solution: Take an appropriate amount of system suitability solution, place it in a suitable volumetric bottle, dilute it 100-200 times with methanol, shake well, and obtain the sensitivity solution.

[0016] Step 3, preparation of the test solution: take 1 ml of irinotecan liposome injection, dilute it 20-100 times with methanol, sonicate, cool, shake well, centrifuge, and take the supernatant;

[0017] Step 4, determination: Take 5-50 μl of the system suitability solution, sensitivity solution, and test solution, inject them into a high performance liquid chromatograph to obtain a chromatogram, and calculate the content of each impurity in the test solution by the area normalization method based on the peak area of ​​the chromatogram.

[0018] The chromatographic conditions are as follows:

[0019] HPLC: VWD or DAD

[0020] Chromatographic column: octadecylsilane bonded silica gel as filler Mobile phase: Mobile phase A Sodium hexane sulfonate solution (take 2g of 1-sodium hexane sulfonate, dissolve in water and dilute to 1000ml): Mobile phase B Acetonitrile Flow rate: 1.0ml / min; Detection wavelength: 254nm, injection volume 5-50μl; Column temperature 30℃;

[0021] Gradient elution

[0022]

[0023]

[0024] The method of the present invention is most preferably carried out in the following steps:

[0025] Step 1, system suitability solution preparation:

[0026] Take irinotecan hydrochloride reference substance and impurity E reference substance, weigh them, add methanol to dissolve them and dilute them into a solution containing 200 μg irinotecan hydrochloride and 0.4 μg impurity E per 1 ml.

[0027] Step 2, preparation of sensitivity solution:

[0028] Take an appropriate amount of system suitability solution, place it in a suitable volumetric bottle, dilute it 100 times with methanol, shake well, and obtain the sensitivity solution.

[0029] Step 3, preparation of test solution:

[0030] Take 1 ml of irinotecan liposome injection, place it in a 25 ml volumetric flask, add methanol and ultrasonicate for 5 minutes, let it cool, then add methanol to dilute to the scale, shake well, centrifuge at 10,000 rpm for 10 minutes, and the supernatant is the test solution.

[0031] Wherein step 4 is to determine:

[0032] Take 25 μl of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph to obtain the chromatogram, and calculate the content of each impurity in the test solution according to the peak area of ​​the chromatogram and the area normalization method.

[0033] General requirements for the detection process: the blank solution (methanol) has no interference; the separation degree of irinotecan and impurity E in the system suitability solution chromatogram is not less than 4.0; in the sensitivity solution chromatogram, the signal-to-noise ratio of the irinotecan peak should be greater than 10.

[0034] The differences between the chromatographic conditions of the present invention and the prior art are:

[0035] The prior art generally adopts HPLC isocratic elution method and UV-visible spectrophotometry. The differences between the chromatographic conditions of the present invention and the prior art are as follows:

[0036] The chromatographic conditions of the present invention adopt the HPLC gradient elution method. The application of the gradient elution method can improve the peak shape of the chromatographic peak, detect multiple impurities at the same time, improve the separation ability, improve the sensitivity, and improve the accuracy and reliability of detection.

[0037] The existing technology uses HPLC isocratic elution chromatography conditions, which has the disadvantage of only being able to detect one component at a time.

[0038] Compared with the prior art, the detection method of the present invention has the following advantages in terms of technical indicators and detection data:

[0039] (1) The present invention adopts the area normalization method of HPLC-gradient elution to detect related substances in irinotecan hydrochloride liposome injection, which has the following advantages:

[0040] ① One detection method can detect multiple impurities (camptothecin, impurity A, impurity B and impurity E) at the same time, and the detection efficiency is high.

[0041] ② The HPLC-gradient elution method has good specificity (peak purity reaches 999), that is, there is no interference between blank, excipient blank and irinotecan, and the separation between impurities is good.

[0042] ③ The HPLC-gradient elution method is used, which has high sensitivity, strong response of each impurity peak and good peak shape.

[0043] ④Simple sample pretreatment.

[0044] ⑤The dilution solvent is methanol, which is cheap and easily available.

[0045] ⑥Agilent conventional chromatographic columns are easily available and inexpensive.

[0046] (2) The test data of the detection method of the present invention are as follows:

[0047] After deducting the blank peak, if there is an impurity peak, calculate the impurity content by the area normalization method.

[0048]

[0049]

[0050] Note: A is the peak area of ​​each impurity, A 总峰 It is the total peak area after correction of each impurity in the test solution. F is the relative response factor of each impurity. The default value of F for unknown impurities is 1. Impurities less than 0.05% are ignored but included in the total impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 blank solution

[0052] Figure 2 Blank excipient solution

[0053] Figure 3 System Suitability Solutions

[0054] Figure 4 Sensitivity solution

[0055] Figure 5 Test solution DETAILED DESCRIPTION

[0056] The present invention is further illustrated by the following examples, but are not intended to limit the present invention.

[0057] Example 1

[0058] The related substances of irinotecan hydrochloride liposome injection were determined by the following method.

[0059] 1) Chromatographic conditions:

[0060]

[0061]

[0062] 2) Solution preparation:

[0063]

[0064]

[0065] 3) Determination: Take 25 μl of blank solution, system suitability solution, sensitivity solution, blank excipient solution and test solution, inject into high performance liquid chromatograph to obtain chromatogram, calculate the related substances of irinotecan hydrochloride liposome injection in the test solution according to the peak area of ​​the chromatogram and the area normalization method, and the results are as follows.

[0066] Camptothecin was not detected, impurity E: not detected, camptothecin: not detected, impurity A: not detected, impurity B: not detected, other maximum single unknown impurity 0.05%, total impurities: 0.10%.

[0067] Example 2

[0068] Screening of chromatographic conditions of the present invention:

[0069] The flow rate conditions were 0.8-1.2 ml / min and the column temperature conditions were 20-60°C. The results are as follows:

[0070] After screening of flow rate and column temperature conditions, it was found that when the flow rate was 1.0 ml / min and the column temperature was 55°C, irinotecan hydrochloride had good separation from camptothecin, impurity A, impurity B, and impurity E, and there was no interference with the blank and excipient blank.

[0071] Example 3

[0072] Test results of irinotecan liposome injection produced from one batch:

[0073] Step 1, system suitability solution preparation: The method is as follows:

[0074] Take irinotecan hydrochloride reference substance and impurity E reference substance, weigh them, add diluent to dissolve them and dilute them into a solution containing 200 μg irinotecan hydrochloride and 0.4 μg impurity E per 1 ml.

[0075] Step 2, sensitivity solution: accurately measure 1 ml of system suitability solution, place it in a 200 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0076] Step 3, preparation of test solution: The method is as follows:

[0077] Take 1 ml of irinotecan liposome injection, place it in a 25 ml brown volumetric bottle, add methanol and ultrasonicate for 5 minutes, let it cool, then add methanol to dilute to the scale, shake well, centrifuge at 10,000 rpm for 10 minutes, and the supernatant is the test solution.

[0078] Step 3, determination, the method is as follows:

[0079] Take 25 μl of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph to obtain the chromatogram, and calculate the content of each impurity in the test solution according to the peak area of ​​the chromatogram. The results are as follows.

[0080] Camptothecin was not detected, impurity E: not detected, camptothecin: not detected, impurity A: not detected, impurity B: not detected, other maximum single unknown impurity 0.05%, total impurities: 0.05%.

[0081] Compared with the prior art, the present invention has the following advantages:

[0082] (1) The present invention uses a high performance liquid chromatography ultraviolet detection method and a gradient elution method to determine the related substances in irinotecan hydrochloride liposome injection according to the area normalization method.

[0083] (2) The diluent of the present invention is methanol, which is cheap and easily available.

[0084] (3) The sample pretreatment of the present invention is simple and the operation is fast.

[0085] (4) The sample detection method of the present invention adopts area normalization, which is time-saving and has high inspection efficiency.

Claims

1. A method for determining related substances in irinotecan hydrochloride liposome injection, the method comprising the following steps: Step 1, System Suitability Solution Preparation Step 2, preparation of sensitivity solution Step 3, preparation of test solution Step 4, determination: take the system suitability solution, the sensitivity solution and the test solution, inject them into the high performance liquid chromatograph to obtain a chromatogram, and calculate the content of impurities in the test solution according to the peak area of ​​the chromatogram by the area normalization method; The chromatographic conditions are as follows: HPLC: VWD or DAD Chromatographic column: Octadecylsilane bonded silica gel as filler Mobile phase: Mobile phase A sodium hexane sulfonate solution (take 2g of 1-sodium hexane sulfonate, dissolve it in water and dilute it to 1000ml): Mobile phase B acetonitrile Flow rate: 1.0ml / min; Detection wavelength: 254nm, injection volume 5-50ul; Column temperature 30℃; Gradient elution 2. The method according to claim 1, characterized in that Step 1, preparation of system suitability solution: the method is as follows: Take irinotecan hydrochloride reference substance and impurity E reference substance, add methanol to prepare solutions containing 100-300 μg of irinotecan hydrochloride and 0.1-0.5 μg of impurity E per 1 ml.

3. The method according to claim 1, characterized in that: Step 2, preparation of sensitivity solution: the method is as follows: Take an appropriate amount of system suitability solution, place it in a suitable volumetric bottle, dilute it 100-200 times with methanol, shake well, and obtain the sensitivity solution.

4. The method according to claim 1, characterized in that: Step 3, preparation of test solution: the method is as follows: Take 1 ml of irinotecan liposome injection, dilute it 20-100 times with methanol, sonicate, cool, shake well, centrifuge, and take the supernatant.

5. The method according to claim 1, characterized in that Wherein step 4, determination, method is as follows: Take 5-50 μl of system suitability solution, sensitivity solution and test solution, inject them into high performance liquid chromatograph to obtain chromatogram, and calculate the content of each impurity in the test solution by area normalization method according to the peak area of ​​chromatogram.

6. The method according to claim 1, characterized in that Here are the steps: Step 1, system suitability solution preparation: The method is as follows: Take irinotecan hydrochloride reference substance and impurity E reference substance, weigh them, add diluent to dissolve and dilute to a solution containing 200 μg irinotecan hydrochloride and 0.4 μg impurity E per 1 ml; Step 2, sensitivity solution: accurately measure 1 ml of system suitability solution, place it in a 200 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain; Step 3, preparation of test solution: The method is as follows: Take 1 ml of irinotecan liposome injection, place it in a 25 ml brown volumetric bottle, add methanol and sonicate for 5 minutes, let it cool, then add methanol to dilute to the scale, shake well, centrifuge at 10,000 rpm for 10 minutes, and the supernatant is the test solution; Step 3, determination, the method is as follows: Take 25 μl of the reference solution and the test solution, inject them into the high performance liquid chromatograph to obtain the chromatogram, and calculate the content of each impurity in the test solution according to the peak area of ​​the chromatogram.

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