Detection method for injection encapsulation efficiency of cytomegalovirus inhibitor

Abstract

The invention relates to a method for detecting the encapsulation efficiency of an injection of a cytomegalovirus inhibitor. Comprising the following steps: mixing an injection of the cytomegalovirus inhibitor with diethyl ether according to a certain volume ratio, carrying out extraction separation, and carrying out quantitative analysis by using high performance liquid chromatography after separation. The detection method is simple and rapid, and can ensure the accuracy of the detection result of the encapsulation efficiency without using a complex instrument and almost without damaging the structure of the inclusion compound. Meanwhile, the method meets the requirements of accuracy and repeatability.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting the encapsulation rate of an injection of a cytomegalovirus inhibitor. Background Art

[0002] Letermovir is a new type of non-nucleoside cytomegalovirus (CMV) inhibitor, which inhibits the activity of the CMV terminal enzyme complex and prevents the processing and packaging of viral DNA, thereby exerting its antiviral effect. The original developer of Letermovir tablets and injection is Merck, which is the world's first and currently the only drug approved for the prevention of CMV infection in allogeneic hematopoietic stem cell transplant recipients. It lays the foundation for the preventive treatment model of CMV and provides doctors with a new option to solve CMV-related problems in the process of hematopoietic stem cell transplantation and improve the overall treatment effect of patients.

[0003] Letermovir injection is composed of Letermovir, hydroxypropyl beta-cyclodextrin, sodium chloride, sodium hydroxide, and water for injection. CN202310163770.7 points out that Letermovir is included in hydroxypropyl beta-cyclodextrin to form a Letermovir inclusion complex. Letermovir needs to be released from the cyclodextrin inclusion complex molecules into the blood in vivo to exert its efficacy. The encapsulation rate will greatly affect the efficacy of the drug in vivo. Therefore, providing a method for accurately detecting the encapsulation rate is of great significance to the quality control of Letermovir injection.

[0004] Therefore, there is an urgent need in the art for a low-cost, simple-to-operate, and accurate method for detecting the encapsulation efficiency of letermovir injection. Summary of the invention

[0005] Based on the above technical problems, the present invention provides a method for detecting the encapsulation rate of Letermovir injection, which is simple to operate, efficient, does not require the use of complex instruments, and is suitable for use.

[0006] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0007] In one aspect, the present invention provides a method for detecting the encapsulation efficiency of letermovir injection, comprising the steps of:

[0008] (s1) mixing a sample to be tested with a weakly polar organic solvent, allowing the mixture to stand, and taking an organic phase A, wherein the sample to be tested comprises Letermovir injection;

[0009] (s2) diluting the organic phase A with methanol to obtain a test solution; and

[0010] (s3) using high performance liquid chromatography to detect the test solution, calculating the content of free letermovir in the test sample, thereby obtaining the encapsulation efficiency of the letermovir injection of the test sample;

[0011] Wherein, the weakly polar organic solvent is selected from the following group: ether, ethyl acetate, dichloromethane or acetone.

[0012] In another preferred embodiment, the Letermovir injection comprises Letermovir, hydroxypropyl-β-cyclodextrin, sodium hydroxide, and sodium chloride.

[0013] In another preferred embodiment, each 1 mL of the Letermovir injection comprises: 20 mg of Letermovir, 145 mg to 155 mg of hydroxypropyl-β-cyclodextrin and 3 mg to 3.5 mg of sodium chloride; preferably, each 1 mL of the Letermovir injection comprises: 20 mg of Letermovir, 150 mg of hydroxypropyl-β-cyclodextrin and 3.1 mg of sodium chloride; the Letermovir injection also comprises an appropriate amount of sodium hydroxide.

[0014] In another preferred embodiment, the specifications of the Letermovir injection are 12mL: 240mg and 24mL: 480mg.

[0015] In another preferred embodiment, the weakly polar organic solvent is diethyl ether.

[0016] In another preferred embodiment, the volume ratio of the sample to be tested to the weakly polar organic solvent is 1:1-100, preferably 1:1-50, more preferably 1:1-20, more preferably 1:1-10, for example, about 1:9, 1:4, 3:7, 4:6, or 1:1.

[0017] In another preferred embodiment, in step (s2), the organic phase A is diluted 1 to 20 times, preferably 3 to 15 times, and more preferably 5 to 12 times.

[0018] In another preferred embodiment, the content of free Letermovir in the sample to be tested is obtained by using an external standard method, so as to further calculate the encapsulation efficiency of the Letermovir injection.

[0019] In another preferred embodiment, in the high performance liquid chromatography detection, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a mixture of potassium dihydrogen phosphate solution and acetonitrile, and the mobile phase B is acetonitrile.

[0020] In another preferred embodiment, the pH of the potassium dihydrogen phosphate solution is 4-8, preferably 4-7, more preferably 4-6, for example, about 5.

[0021] In another preferred embodiment, the mobile phase A is 12 mmol / L potassium dihydrogen phosphate solution-acetonitrile (9:1).

[0022] In another preferred embodiment, in the HPLC detection, a C18 chromatographic column is used, preferably Halo C18 4.6 mm×150 mm 2.7 μm; and octadecylsilane bonded silica gel is used as a filler.

[0023] In another preferred embodiment, in the HPLC detection, the flow rate is 0.1-2 ml / min, preferably 0.3-1 ml / min, more preferably 0.5-1 ml / min, for example, about 0.8 ml / min.

[0024] In another preferred embodiment, in the HPLC detection, the column temperature is 40-50°C, preferably 45°C.

[0025] In another preferred embodiment, in the HPLC detection, the detection wavelength is 200-250 nm, preferably 210-240 nm, more preferably 210-230 nm, for example, about 220 nm.

[0026] In another preferred embodiment, in the HPLC detection, the injection volume is 10 μl.

[0027] In another preferred embodiment, the method further comprises the detection of a reference substance, comprising the steps of:

[0028] (1) using methanol to dissolve and dilute a reference substance to obtain a reference substance solution C, wherein the reference substance is letermovir; and

[0029] (2) Using high performance liquid chromatography to detect the reference solution C, and obtaining a high performance liquid chromatography detection result of the reference solution.

[0030] In another preferred embodiment, the method further comprises the detection of a reference substance, comprising the steps of:

[0031] (1) Dissolve 10-20 mg of the reference substance in 20-30 ml of methanol to obtain reference substance solution B;

[0032] (2) diluting 1 to 5 ml of reference solution B to 10 to 40 ml using methanol to obtain reference solution C; and

[0033] (3) Using high performance liquid chromatography to detect the reference solution C, and obtaining a high performance liquid chromatography detection result of the reference solution.

[0034] In another preferred embodiment, the encapsulation efficiency of the Letermovir injection is calculated by formula (I):

[0035] Encapsulation efficiency % = 100% - free letermovir content % (I).

[0036] In another preferred embodiment, the free letermovir content is calculated by the external standard method.

[0037] In another preferred embodiment, the free letermovir content is calculated by formula (II):

[0038] Free letermovir content % = (A s / A r *C r *n)*V s2 / (C s *V s1 )*100%(II)

[0039] In the formula, A s : Letermovir peak area in the test solution;

[0040] A r : Letermovir peak area in reference solution C;

[0041] C r : Letermovir concentration in reference solution C, mg / ml;

[0042] n: dilution factor of organic phase A;

[0043] V s1 : The volume of the sample to be tested;

[0044] C s : The theoretical concentration of letermovir in the sample to be tested;

[0045] V s2 : The volume of weakly polar organic solvent.

[0046] In another preferred embodiment, in the high performance liquid chromatography detection, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a mixture of potassium dihydrogen phosphate solution and acetonitrile, and the mobile phase B is acetonitrile.

[0047] In another preferred embodiment, in the HPLC detection, the elution gradient is shown in the following table

[0048]

[0049] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0050] (1) The present invention uses a certain volume ratio of ether to extract the letermovir injection, which can not only effectively separate the letermovir inclusion complex from the free letermovir, but also ensure that the structure of the inclusion complex is not destroyed during the determination process, thereby avoiding leakage of the encapsulated drug and causing a falsely low encapsulation rate.

[0051] (2) The present invention provides a simple and efficient method for detecting the encapsulation efficiency of letermovir injection, which does not require the use of complex instruments and meets the requirements of accuracy and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The HPLC chromatogram of the test solution extracted according to Ratio 1 in Example 3 is shown in FIG.

[0053] Figure 2 The HPLC chromatogram of the test solution extracted according to the ratio 2 in Example 3 is shown in FIG.

[0054] Figure 3 The figure is a high performance liquid chromatogram of the test solution extracted according to the ratio 3 in Example 3.

[0055] Figure 4 The HPLC chromatogram of the test solution extracted according to the ratio 4 in Example 3 is shown in FIG.

[0056] Figure 5 The HPLC chromatogram of the test solution extracted according to the ratio of 5 in Example 3 is shown in FIG.

[0057] Figure 6 The HPLC chromatogram of the test solution extracted according to the ratio 6 in Example 3 is shown in FIG.

[0058] Figure 7 The HPLC chromatogram of the test solution extracted according to the ratio 7 in Example 3 is shown in FIG.

[0059] Figure 8 It is the HPLC chromatogram of the reference substance solution in Example 3. DETAILED DESCRIPTION

[0060] The inventors first investigated the applicability of the commonly used ultrafiltration centrifugation method, and found that the inclusion complex of Letermovir may pass through the ultrafiltration membrane under the action of centrifugal force, resulting in a decrease in the encapsulation rate. Subsequently, different weakly polar solvents were screened to extract free Letermovir. The weakly polar organic solvent and the inclusion complex of Letermovir have a large polarity difference and are immiscible, thereby achieving the purpose of separating free Letermovir from the inclusion complex of Letermovir.

[0061] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0062] Unless otherwise specified, the percentages of substance concentration or content in the present invention are expressed as weight percentage.

[0063] The relevant materials used in the examples are as follows:

[0064] Letermovir API, source: Shanghai Desano Chemical Pharmaceutical Co., Ltd., batch number: DVB373-X230807 Letermovir reference substance, source: Allite (Shanghai) Pharmaceutical Technology Co., Ltd., batch number: D373RWSA2201

[0065] Letermovir injection, source: homemade in the laboratory, specification: 12ml / bottle, the prescription is exactly the same as the reference preparation (trade name: PREVYMIS), the specific prescription is as follows: Letermovir 240mg / bottle, hydroxypropyl beta-cyclodextrin 1800mg / bottle, sodium chloride 37.2mg / bottle, appropriate amount of sodium hydroxide (pH adjuster).

[0066] High performance liquid chromatograph, source: Waters, model: Arc 2489

[0067] Chromatographic column, source: Halo, model: C18, specification: 4.6mm×150mm, 2.7μm, serial number: USFH009728

[0068] Example 1

[0069] Ultrafiltration centrifugation study

[0070] The molecular weight of the Letermovir inclusion complex is about 2000, and the molecular weight of the free Letermovir is 572.6. Based on the difference in molecular weight between the inclusion complex and the free state, an ultrafiltration tube with a molecular retention capacity of 1000 is selected.

[0071] (1) Free letermovir permeability test

[0072] Letermovir solution (1 mg / ml): Accurately weigh 49.50 mg of Letermovir API and 7.67 mg of sodium chloride, place in the same 50 ml volumetric flask, add about 40 ml of 70% ethanol solution to dissolve, transfer to a beaker, adjust the pH to 7.503 with 0.25 M sodium hydroxide solution, and then completely transfer to a 50 ml volumetric flask, dilute to the scale with 70% ethanol solution, and shake well.

[0073] Letermovir solution (2 mg / ml): Accurately weigh 99.15 mg of Letermovir API and 15.34 mg of sodium chloride, place in the same 50 ml volumetric flask, add about 40 ml of 70% ethanol solution to dissolve, transfer completely to a beaker, adjust the pH to 7.546 with 0.25 M sodium hydroxide, transfer completely to a 50 ml volumetric flask, dilute to the mark with 70% ethanol solution, and shake well.

[0074] Letermovir solution (3 mg / ml): Accurately weigh 149.54 mg of Letermovir API and 23.15 mg of sodium chloride, place in the same 50 ml volumetric flask, add about 40 ml of 70% ethanol solution to dissolve, transfer completely to a beaker, adjust the pH to 7.527 with 0.25 M sodium hydroxide, transfer completely to a 50 ml volumetric flask, dilute to the mark with 70% ethanol solution, and shake well.

[0075] Ultrafiltration solution 1: Accurately measure 4 ml of letermovir solution (1 mg / ml) in an ultrafiltration tube, place the ultrafiltration tube in a centrifuge and centrifuge (6800 rpm) for 5, 10, and 20 minutes, discard the ultrafiltration solution in the receiving tube after the first two centrifugations, and take the filtrate from the third centrifugation. Accurately measure 0.5 ml of the ultrafiltration solution, place it in a 25 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0076] Ultrafiltration solution 2: Accurately measure 4 ml of letermovir solution (2 mg / ml) in an ultrafiltration tube, place the ultrafiltration tube in a centrifuge for centrifugation (6800 rpm), centrifuge for 5 minutes, 10 minutes, and 20 minutes, discard the ultrafiltration solution in the receiving tube after the first two centrifugations, and take the filtrate from the third centrifugation. Accurately measure 0.5 ml of the ultrafiltration solution, place it in a 25 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0077] Ultrafiltration solution 3: Accurately measure 4 ml of letermovir solution (3 mg / ml) in an ultrafiltration tube, place the ultrafiltration tube in a centrifuge and centrifuge (6800 rpm) for 5, 10, and 20 minutes, discard the ultrafiltration solution in the receiving tube after the first two centrifugations, and take the filtrate from the third centrifugation. Accurately measure 0.5 ml of the ultrafiltration solution, place it in a 25 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0078] Letermovir reference solution: Accurately weigh 12.56 mg of Letermovir reference, place in a 25 ml volumetric flask, add methanol to dissolve and dilute to scale, shake well. Accurately measure 2 ml of the above solution, place in a 25 ml volumetric flask, dilute to scale with methanol, shake well.

[0079] The letermovir reference solution, ultrafiltration solution 1, ultrafiltration solution 2, and ultrafiltration solution 3 were analyzed by high performance liquid chromatography, and the letermovir concentration in the ultrafiltration solution was calculated according to the external standard method. The chromatographic conditions were as follows: octadecylsilane bonded silica gel as filler, mobile phase A: 12 mmol / L potassium dihydrogen phosphate solution (pH = 4.9)-acetonitrile (9:1), mobile phase B: acetonitrile, flow rate: 0.8 ml / min, column temperature: 45 ° C, detection wavelength: 220 nm, injection volume: 10 μl, and the elution gradient was as follows:

[0080]

[0081] Recovery rate calculation formula: Recovery rate (%) = measured amount / added amount * 100%

[0082] If the recovery rate can reach more than 98%, it means that free letermovir can effectively pass through the ultrafiltration membrane, while the letermovir inclusion complex is retained, thus achieving the separation of the two. The test results are as follows, and the recovery rates are all higher than 98%, indicating that free letermovir can completely pass through the ultrafiltration membrane under the action of centrifugal force.

[0083]

[0084] (2) Ultrafiltration centrifugation method to detect the encapsulation efficiency of letermovir injection

[0085] Test solution: Measure 4 ml of letermovir injection and place it in an ultrafiltration tube. Place the ultrafiltration tube in a centrifuge and centrifuge it 3 times (6800 rpm). The centrifugation time is 5 minutes, 10 minutes, and 20 minutes respectively. Discard the ultrafiltration solution in the receiving tube after the first two centrifugations and take the filtrate from the third centrifugation. Accurately measure 0.5 ml of the filtrate and place it in a 50 ml volumetric flask. Dilute it to the mark with methanol and shake well. Prepare 6 test solutions in parallel.

[0086] Letermovir reference solution: accurately weigh 12.68 mg of Letermovir reference, place in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well; accurately measure 2 ml of the above solution, place in a 25 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0087] The letermovir reference solution and test solutions 1 to 6 were analyzed by HPLC. The HPLC method was the same as above. The free letermovir content in the six test solutions was calculated by the external standard method, and the encapsulation efficiency was calculated.

[0088] The formula for calculating the encapsulation efficiency of ultrafiltration centrifugation is as follows:

[0089] The encapsulation rate calculation formula is: encapsulation rate % = 100% - free letermovir content %.

[0090] Free letermovir content % = (A s / A r *C r *V s ) / C s *100%.

[0091] Where:

[0092] A s : Letermovir peak area in the test solution;

[0093] A r : Letermovir peak area in reference solution;

[0094] C r : Letermovir concentration in reference solution, mg / ml;

[0095] V s : dilution multiple of the test solution;

[0096] C s : Theoretical concentration of the test solution, 20 mg / ml.

[0097] The test results are as follows:

[0098] Solution name Free letermovir content% Encapsulation rate% Test solution 1 8.53 91.5 Test solution 2 8.37 91.6 Test solution 3 23.06 76.9 Test solution 4 7.14 92.9 Test solution 5 6.09 93.9 Test solution 6 5.97 94.0 Average value % (n=6) N / A 90.1 RSD% (n=6) N / A 7.3

[0099] Note: N / A means not applicable.

[0100] With reference to the Chinese Pharmacopoeia 2020 Edition Part IV and ICH related guidelines, the main component content determination requires that the RSD of the content of 6 test sample solutions in the repeatability test shall not be greater than 2.0%. The RSD of the encapsulation efficiency of 6 Letermovir injections in the present invention is 7.3% (n=6), and the repeatability does not meet the requirements. The inventor speculates that the Letermovir inclusion complex may pass through the ultrafiltration membrane under the action of centrifugal force, resulting in a decrease in the encapsulation efficiency, so the ultrafiltration centrifugation method is not applicable.

[0101] Example 2

[0102] Extraction method investigation

[0103] According to the polarity characteristics of the compounds, since the polarity of Letermovir is small and the polarity of hydroxypropyl-beta-cyclodextrin and the inclusion complex is large, the inventors used the principle of like dissolves like and used a weakly polar organic solvent to extract free Letermovir, thereby achieving the purpose of separating free Letermovir from the Letermovir inclusion complex.

[0104] In this regard, the inventors screened different weakly polar solvents, including ethyl acetate, dichloromethane, acetone and diethyl ether, and added them to the letermovir injection (i.e., the sample) in a quantitative manner. The content of free letermovir in the extraction solution was determined by high performance liquid chromatography, and the encapsulation efficiency was calculated. The high performance liquid chromatography method was the same as that in Example 1.

[0105] The formula for calculating the encapsulation efficiency of the extraction method is as follows:

[0106] Encapsulation efficiency % = 100% - free letermovir content %;

[0107] Free letermovir content % = (A s / A r *C r *10)*V s2 / (C s *V s1 )*100%

[0108] Where:

[0109] A s : Letermovir peak area in the test solution;

[0110] A r : Letermovir peak area in reference solution C;

[0111] C r : Letermovir concentration in reference solution, mg / ml;

[0112] 10: dilution factor of the weak polar solvent used in the preparation of the test solution;

[0113] V s1 : The volume of sample used during extraction, ml.

[0114] C s : Theoretical concentration of the sample, 20 mg / ml;

[0115] V s2 : Volume of weak polar solvent used during extraction, ml.

[0116] The results are as follows:

[0117] Extraction solvent Free letermovir content% Encapsulation rate% Ethyl acetate (1 ml sample and 9 ml ethyl acetate) 35.7 64.3 Dichloromethane (1 ml sample and 9 ml dichloromethane) 23.4 76.6 Acetone (1 ml sample and 9 ml acetone) 43.4 56.6 Ether (1 ml sample and 9 ml ether) 7.9 92.1

[0118] Weakly polar organic solvents such as ethyl acetate, dichloromethane, acetone and ether all have certain extraction and separation effects on free letermovir, and the encapsulation rate of extraction with ether is the highest. Analysis of the reasons may be that ethyl acetate, dichloromethane and acetone destroy the inclusion state and cause letermovir to be free. In summary, ether was selected as the extraction solvent.

[0119] Example 3

[0120] Effect of volume ratio of ether on encapsulation efficiency

[0121] In this embodiment, 7 groups of solvents with different volume ratios were set for extraction. After the extraction was completed, the free letermovir content was determined by high performance liquid chromatography, and the steps were as follows:

[0122] Step 1: Accurately measure the letermovir injection (i.e., sample), place it in a volumetric flask, dilute to the mark with ether, and shake manually to mix.

[0123] Step 2: Let stand until the upper organic phase is clear, accurately measure 3 ml of the upper organic phase and place it in a 20 ml volumetric flask, and dilute it with methanol to obtain the test solution.

[0124] Step 3: Accurately weigh 15.47 mg of the Letermovir reference substance, place it in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, and shake well; Accurately measure 3 ml of the above solution, place it in a 20 ml volumetric flask, dilute to the scale with methanol, and shake well to obtain the Letermovir reference substance solution.

[0125] Step 4: Accurately measure the test solution and the letermovir reference solution, detect them by high performance liquid chromatography, calculate the content of free letermovir by external standard method, and calculate the encapsulation efficiency by the same analysis method as in Example 1.

[0126] The HPLC chromatogram of the test solution is shown in Figure 1-7 , the HPLC chromatogram of the reference solution is shown in Figure 8 The calculated encapsulation efficiency results are shown in the table below. The encapsulation efficiency of ratios 1 and 2 is significantly lower than that of ratios 3 to 7. There is no significant difference in the encapsulation efficiency of ratios 3 to 7. Therefore, the volume ratios of sample: ether of 1:9, 2:8, 3:7, 4:6 and 5:5 can achieve good extraction effects. In subsequent experiments, the ratio 5 (sample: ether = 3:7) with the highest encapsulation efficiency was selected for extraction.

[0127] name Preparation method Free letermovir content% Encapsulation rate% Scale 1 0.5 ml sample - 49.5 ml ether 13.8 86.2 Scale 2 0.5 ml sample - 24.5 ml ether 14.7 85.3 Scale 3 1ml sample - 9ml ether 7.9 91.1 Scale 4 2ml sample-8ml ether 7.4 90.6 Scale 5 3ml sample - 7ml ether 6.7 93.3 Scale 6 4ml sample - 6ml ether 7.3 90.1 Ratio 7 5ml sample-5ml ether 6.9 90.4

[0128] Example 4

[0129] Methodological validation

[0130] 4.1 Accuracy test

[0131] Since letermovir is insoluble in water but soluble in a pH 2.0 hydrochloric acid aqueous solution, free letermovir solutions with concentration gradients of 90%, 100%, and 105% were prepared using a pH 2.0 hydrochloric acid aqueous solution, and extraction was performed at a volume ratio of sample: ether = 3:7 to examine the recovery rate.

[0132] 90%, 100%, 105% accuracy solution preparation:

[0133] 1) 90% accuracy solution: Accurately weigh 25.2 mg of Letermovir API, place in a 20 ml volumetric flask, accurately measure and add 0.5 ml of sodium chloride solution, add an appropriate amount of pH 2.0 hydrochloric acid aqueous solution, ultrasonically dissolve, dilute with pH 2.0 hydrochloric acid aqueous solution, and shake well. Immediately accurately measure 3 ml of the above solution, place in a 10 ml volumetric flask, dilute with ether and shake thoroughly, let stand to separate, accurately measure 3 ml of the supernatant, place in a 20 ml volumetric flask, dilute with methanol, and shake well. Prepare 3 copies in parallel.

[0134] 2) 100% accuracy solution: Accurately weigh 28.0 mg of Letermovir API, place in a 20 ml volumetric flask, accurately measure and add 0.5 ml of sodium chloride solution, add an appropriate amount of pH 2.0 hydrochloric acid aqueous solution, ultrasonically dissolve, dilute with pH 2.0 hydrochloric acid aqueous solution, and shake well. Immediately accurately measure 3 ml of the above solution, place in a 10 ml volumetric flask, dilute with ether and shake thoroughly, let stand to separate, accurately measure 3 ml of the supernatant, place in a 20 ml volumetric flask, dilute with methanol, and shake well. Prepare 3 copies in parallel.

[0135] 3) 105% accuracy solution: Accurately weigh 29.4 mg of Letermovir API, place in a 20 ml volumetric flask, accurately measure and add 0.5 ml of sodium chloride solution, add an appropriate amount of pH 2.0 hydrochloric acid aqueous solution, ultrasonically dissolve, dilute with pH 2.0 hydrochloric acid aqueous solution, and shake well. Immediately accurately measure 3 ml of the above solution, place in a 10 ml volumetric flask, dilute with ether and shake thoroughly, let stand to separate, accurately measure 3 ml of the supernatant, place in a 20 ml volumetric flask, dilute with methanol, and shake well. Prepare 3 copies in parallel.

[0136] Letermovir reference solution: accurately weigh 15.65 mg of Letermovir reference, place in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well; accurately measure 3 ml of the above solution, place in a 20 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0137] The free letermovir content was analyzed by high performance liquid chromatography. The analysis method was the same as in Example 1. The letermovir reference solution, 90% accuracy solution, 100% accuracy solution, and 105% accuracy solution were sampled and analyzed respectively. The letermovir content was determined by the external standard method, and the recovery rate was calculated. The calculation formula was: recovery rate (%) = measured amount / added amount * 100%. The results are as follows:

[0138]

[0139] At different concentrations of free drugs, the recovery rate was between 97.53% and 102.22%, which met the requirements; the RSD (n = 3) was 1.9%, 1.7%, and 1.2%, respectively, and the RSD (n = 9) was 1.6%, all less than 2.0%, which met the requirements. This shows that the method can extract completely at different concentrations of free drugs, and the accuracy of the method is good.

[0140] 4.2 Repeatability test

[0141] Letermovir reference solution: accurately weigh 15.39 mg of Letermovir reference, place in a 25 ml volumetric flask, add methanol to dissolve and dilute to the mark, shake well; accurately measure 3 ml of the above solution, place in a 20 ml volumetric flask, dilute to the mark with methanol, shake well.

[0142] Repeatable test solutions 1 to 6: Prepare the test solution in the same way, i.e. accurately measure 3 ml of letermovir injection (prepared in the laboratory), place it in a 10 ml volumetric flask, dilute to the mark with ether, and mix by manual oscillation; let it stand until the upper organic phase becomes clear, accurately measure 3 ml of the upper organic phase and place it in a 20 ml volumetric flask, dilute to the mark with methanol, and prepare 6 test solutions in parallel.

[0143] The free letermovir content was calculated by external standard method, and the encapsulation efficiency was calculated. The analysis method was the same as in Example 1. The free letermovir content of 6 replicate test solutions was measured, and the encapsulation efficiency was calculated. The results were as follows:

[0144] Solution name Free letermovir content% Encapsulation rate% Repeatability Test Solution 1 7.1 92.9 Repeatability Test Solution 2 6.9 93.1 Repeatability Test Solution 3 7.3 92.7 Repeatability Test Solution 4 6.7 93.3 Repeatability Test solution 5 7.3 92.6 Repeatability Test Solution 6 7.3 92.6 Average value % (n=6) N / A 92.9 RSD% (n=6) N / A 0.3

[0145] Note: N / A means not applicable.

[0146] The average encapsulation efficiency of Letermovir injection of repeatable test solutions 1 to 6 was 92.9%, and the RSD value was 0.3%, which met the repeatability requirements.

[0147] In summary, the present invention uses a certain volume ratio of ether to extract the letermovir injection, which can effectively separate the free letermovir and the letermovir inclusion complex, and the process is simple and fast, without the need for complex instruments and almost without destroying the inclusion complex structure. At the same time, the method meets the requirements of accuracy and repeatability.

[0148] discuss

[0149] At present, the encapsulation rate detection methods of Letermovir Injection include column chromatography, centrifugation, ultrafiltration centrifugation, etc.

[0150] 1) The separation principle of column chromatography is to separate substances based on their different adsorption capacities on the stationary phase. The inventors have found through actual research that the inclusion complex formed by letermovir and hydroxypropyl-beta-cyclodextrin and the free letermovir are in a dynamic equilibrium process. The inclusion complex is easily destroyed during column chromatography, resulting in a falsely low encapsulation rate. This method is not applicable.

[0151] 2) The centrifugation method is to make liposomes or nanoparticles sink under different centrifugal forces to separate and remove free drugs in different types of liposomes or nanoparticles. To some extent, it also depends on the flocculation state of the suspension. The Letermovir inclusion complex is completely dissolved in the aqueous solution through solubilization with hydroxypropyl-beta-cyclodextrin, so it cannot be separated by centrifugation. Therefore, this method is not applicable.

[0152] 3) Ultrafiltration centrifugation is to place the sample in an ultrafiltration tube equipped with an ultrafiltration membrane and centrifuge at a suitable speed. The free drug can pass through the ultrafiltration membrane under the action of centrifugal force, while the macromolecular inclusion complex is retained, thereby achieving separation of the two. The data of Example 1 of this application show that the repeatability of ultrafiltration centrifugation is low, so this method is not applicable.

[0153] The method of the present invention can accurately extract free Letermovir from Letermovir injection, and use high performance liquid chromatography to calculate the content of free Letermovir, thereby calculating its encapsulation efficiency. The encapsulation efficiency measured by the method of the present invention is accurate, reproducible, simple to operate, low in cost, and has a wide range of applications.

[0154] In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for detecting the encapsulation efficiency of letermovir injection, characterized in that: Includes steps: (s1) mixing a sample to be tested with a weakly polar organic solvent, allowing the mixture to stand, and taking an organic phase A, wherein the sample to be tested comprises Letermovir injection; (s2) diluting the organic phase A with methanol to obtain a test solution; and (s3) using high performance liquid chromatography to detect the test solution, calculating the content of free letermovir in the test sample, thereby obtaining the encapsulation efficiency of the letermovir injection of the test sample; Wherein, the weakly polar organic solvent is selected from the following group: ether, ethyl acetate, dichloromethane or acetone.

2. The detection method according to claim 1, characterized in that The weakly polar organic solvent is ether.

3. The detection method according to claim 1, characterized in that The volume ratio of the sample to be tested to the weakly polar organic solvent is 1:1-100.

4. The detection method according to claim 1, characterized in that The volume ratio of the sample to be tested to the weakly polar organic solvent is 1:1-50.

5. The detection method according to claim 1, characterized in that The volume ratio of the sample to be tested to the weakly polar organic solvent is 1:1-20.

6. The detection method according to claim 1, characterized in that In step (s2), the organic phase A is diluted 1 to 20 times, preferably 3 to 15 times, and more preferably 5 to 12 times.

7. The detection method according to claim 1, characterized in that In the high performance liquid chromatography detection, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a mixture of potassium dihydrogen phosphate solution and acetonitrile, and the mobile phase B is acetonitrile.

8. The detection method according to claim 7, characterized in that The pH of the potassium dihydrogen phosphate solution is 4-8, preferably 4-7, more preferably 4-6, for example, about 5.

9. The detection method according to claim 1, characterized in that: The method also includes the detection of a reference substance, comprising the steps of: (1) using methanol to dissolve and dilute a reference substance to obtain a reference substance solution C, wherein the reference substance is letermovir; and (2) Using high performance liquid chromatography to detect the reference solution C, and obtaining a high performance liquid chromatography detection result of the reference solution.

10. The detection method according to claim 1, characterized in that: In the high performance liquid chromatography detection, a C18 chromatographic column is used, and the filler is octadecylsilane bonded silica gel.

Images