Small molecule detection method in CB-PLG-NPs medicine and application of small molecule detection method

Abstract

The invention relates to the technical field of quality detection, in particular to a method for detecting small molecules in a CB-PLG-NPs drug and application of the method. The detection method provided by the invention can be used for simultaneously detecting the content of two active small molecules BLZ945 and CA4 in the CB-PLG-NPs medicine and the content of two small molecule impurities of 2, 4, 6-trichlorobenzoic acid and an isomer CA4IM01 of CA4, and is high in accuracy and sensitivity and strong in specificity. The detection method disclosed by the invention is applied to the detection of the drug loading capacity of the CB-PLG-NPs drug, is complete and systematic, and is beneficial to really reflecting the loading condition of active small molecules in the drug, really comparing the content of the active small molecules and reducing system errors to the greatest extent so as to ensure that accurate quantification is given during drug administration; and in the whole set of detection method, all related substances are detected, and the detection method can be used as a reference basis for popularization of medicines, and also can be used as a beneficial reference basis for pharmacology and toxicology.

Description

Technical Field

[0001] The invention relates to the technical field of quality detection, and in particular to a small molecule detection method in a CB-PLG-NPs drug and an application thereof. Background Art

[0002] At present, the drugs used to treat advanced liver cancer are mainly multi-kinase inhibitors and PD1 immune drugs, but the efficacy is limited. Advanced liver cancer urgently needs more effective therapeutic drugs. The Compretin A4 (CA4) and BLZ945 co-loaded nanodrug (CB-PLG-NPs) developed by the research and development team of this project is a new type of highly effective systemic therapeutic nanodrug that can be enriched around tumor blood vessels. CA4 selectively destroys tumor blood vessels, causing large-area ischemic necrosis of the tumor. At the same time, the released BLZ945 can reduce the number of M2-TAMs in the tumor, reverse the immunosuppressive microenvironment, and promote CD8+T cell tumor infiltration, thereby achieving efficient synergistic treatment of tumors by CA4 and BLZ945. It has great potential in the field of advanced liver cancer treatment. Its structure is shown in the following formula (1):

[0003]

[0004] At present, the synthesis route of the above-mentioned nanomedicine (CB-PLG-NPs) API is as follows Figure 1 As shown, Figure 1 The schematic diagram of the synthesis route of CB-PLG-NPs raw materials is shown in Figure 2. Figure 1 In the industrial production of the API synthesis route shown, BLZ945 and CA4 were attached to the polymer PLG-A through a Yamaguchi esterification reaction to finally form the CB-PLG-NPs API. The two active small molecules play an important role in the entire drug system. In the synthesis process, the drug loading of the API needs to be strictly controlled. The drug loading, as an important measurement indicator, directly affects the dosage of the nanomedicine and thus the efficacy.

[0005] In view of this, it is necessary to develop an efficient detection method to detect the content of active small molecules CA4 and BLZ945 in nanomedicines, which is of great significance for the control of CB-PLG-NPs process and the characterization of product quality attributes.

[0006] Chinese patent CN 113559275 A discloses a method for preparing CB-PLG-NPs, which includes a drug loading detection. However, the detection method is unable to detect some small molecule impurities, such as CA4 isomers and 2,4,6-trichlorobenzoic acid. In addition, the presence of various impurities and CA4 isomers is ignored in the process of calculating the drug loading, and the obtained results are not accurate. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention lies in a small molecule detection method in CB-PLG-NPs drugs and its application. The small molecule detection method provided by the present invention can simultaneously detect the contents of two active small molecules, BLZ945 and CA4, and two small molecule impurities, 2,4,6-trichlorobenzoic acid and CA4_IM01, the isomer of CA4, in CB-PLG-NPs drugs, with high accuracy and sensitivity and strong specificity.

[0008] The present invention provides a method for detecting small molecules in CB-PLG-NPs drugs, comprising:

[0009] HPLC was used to detect the CB-PLG-NPs drug test solution;

[0010] The chromatographic conditions of the HPLC are:

[0011] A 0.01-0.1 wt% trifluoroacetic acid aqueous solution was used as mobile phase A, a 0.01-0.1 wt% acetonitrile aqueous solution was used as mobile phase B, and gradient elution was performed. The procedure of the gradient elution was as follows:

[0012]

[0013]

[0014] The small molecule detection method in the CB-PLG-NPs drug provided by the present invention uses HPLC to detect the CB-PLG-NPs drug test solution, and the small molecules include BLZ945, CA4, CA4 isomer CA4_IM01 and 2,4,6-trichlorobenzoic acid; the BLZ945 and CA4 exist as active small molecules in the CB-PLG-NPs drug, including free and non-free states; the CA4_IM01 exists as a small molecule impurity in the CB-PLG-NPs drug, including free and non-free states; the 2,4,6-trichlorobenzoic acid exists as a small molecule impurity in the CB-PLG-NPs drug. The CB-PLG-NPs drug of the present invention is the same as that described in the background technology, and will not be repeated. The detection wavelength of the HPLC of the present invention is 205nm~230nm, preferably 220nm. The injection volume of the HPLC of the present invention is 5μL~20μL. The number of injection needles for testing the CB-PLG-NPs drug test solution of the present invention is 1 injection needle for each parallel sample.

[0015] The chromatographic column of HPCL described in the present invention is a reverse phase C 18 Chromatographic column or equivalent chromatographic column, specifically the reverse phase C 18The chromatographic column includes, but is not limited to, a phenylhexyl bonded silica gel chromatographic column, an organic hybrid silica gel chromatographic column, or an octadecylsilane bonded silica gel chromatographic column. 18 The chromatographic column is Waters Xselect CSH Phenyl-Hexyl, with specifications of 4.6×150 mm and 3.5 μm. The column temperature of the HPLC chromatographic column of the present invention is 20° C. to 50° C., preferably 38° C. to 42° C., and more preferably 40° C.

[0016] The present invention uses 0.01-0.1wt% trifluoroacetic acid aqueous solution as mobile phase A, and 0.01-0.1wt% acetonitrile aqueous solution as mobile phase B. In certain embodiments of the present invention, 0.03wt% trifluoroacetic acid aqueous solution is used as mobile phase A, and 0.03wt% acetonitrile aqueous solution is used as mobile phase B. In certain embodiments of the present invention, the flow rate of the mobile phase A and the mobile phase B is 0.5mL / min to 1.6mL / min, preferably 1.4mL / min to 1.6mL / min, and more preferably 1.5mL / min.

[0017] The detection method provided by the present invention can detect the total content of the above-mentioned small molecules in the CB-PLG-NPs drug, and the total content includes both the content of non-free small molecules grafted with CB-PLG-NPs in the CB-PLG-NPs drug and the content of free small molecules in the CB-PLG-NPs drug. Specifically, when the total content of small molecules contained in the CB-PLG-NPs drug test solution is detected by HPLC, the preparation method of the CB-PLG-NPs drug test solution includes: using water, NaOH and acetonitrile as extraction solvents to prepare a CB-PLG-NPs drug test solution. More specifically, water, NaOH and CB-PLG-NPs drugs are mixed, a first ultrasonic extraction is performed, and then the product obtained after the first ultrasonic extraction is mixed with acetonitrile, a second ultrasonic extraction is performed, and then the volume is fixed with a diluent to prepare a CB-PLG-NPs drug test solution; the diluent is a mixed solution of acetonitrile and water in a volume ratio of (1 to 4): 1.

[0018] The detection method provided by the present invention can also detect the content of free small molecules in the CB-PLG-NPs drug separately. Specifically, HPLC is used to detect the free content of small molecules contained in the CB-PLG-NPs drug test solution. The preparation method of the CB-PLG-NPs drug test solution includes: using acetonitrile aqueous solution as an extraction solvent to prepare a CB-PLG-NPs drug test solution. More specifically, the preparation method of the CB-PLG-NPs drug test solution includes: mixing the CB-PLG-NPs drug and acetonitrile aqueous solution to prepare a CB-PLG-NPs drug test solution. In certain embodiments of the present invention, the CB-PLG-NPs drug, 10 mL of acetonitrile and 35 mL of water are mixed to prepare a CB-PLG-NPs drug test solution.

[0019] In order to determine whether the detection method of the present invention meets the acceptance criteria of system suitability, in addition to detecting the CB-PLG-NPs drug test solution, it also includes detecting a blank solution (Blank), a sensitivity solution (LOQ) and a reference solution, wherein the reference solution includes a first reference solution (STD-1) and a second reference solution (STD-2). The blank solution of the present invention is obtained by mixing water, NaOH and acetonitrile, specifically by mixing 25 mL of pure water, 1 mL of 1 mol / L NaOH solution and 20 mL of acetonitrile, cooling to room temperature, adding a diluent to a constant volume of 50 mL, and shaking well to obtain a blank solution. The reference solution of the present invention includes a first reference solution (STD-1) and a second reference solution (STD-2). When the total content or free content of small molecules BLZ945, CA4 and CA4_IM01 in the drug is detected, the preparation method is: 25±2.5 mg of each of the reference substances BLZ945, CA4 and CA4_IM01 is added with a diluent for ultrasonic dissolution and then fixed to 50 mL, 1 mL of the solution obtained after fixed to 10 mL is further fixed to obtain a reference solution; and 2,4,6-trichlorobenzoic acid in the drug only needs to be prepared in the same way when detecting the free content of BLZ945, CA4 and CA4_IM01. The sensitivity solution (LOQ) of the present invention is a solution with a concentration of BLZ945, CA4 and CA4_IM01 of <0.0005 mg / mL, respectively. Specifically, 1 mL of STD-1 solution is accurately pipetted, the diluent is added to the solution to 200 mL, and the solution is shaken to obtain a sensitivity solution. The diluents in the blank solution, sensitivity solution and reference solution are all mixed solutions of acetonitrile and water in a volume ratio of (1 to 4): 1. The present invention detects the blank solution, sensitivity solution and reference solution, and the number of injection needles of the blank solution is ≥ 2, preferably 2 to 5; the number of injection needles of the first reference solution is 5; the rest is the same as the small molecule detection method in CB-PLG-NPs drugs, which will not be repeated.

[0020] The detection method provided by the present invention meets the following system suitability standards:

[0021] (1) The blank solution has no interference at the peak positions of CA4, BLZ945 and CA4_IM01. If there is interference, the peak area of ​​the interference peak shall not be greater than the peak area of ​​each component in the sensitive solution (≤ LOQ), otherwise it should be evaluated.

[0022] (2) The signal-to-noise ratio of CA4_IM01 in the above sensitivity solution should be ≥10.

[0023] (3) The system precision requires that the RSD of the peak area of ​​each component in the chromatogram of the first reference solution injected continuously with 5 injections is ≤5.0%, and the RSD of the retention time is ≤1.0%.

[0024] (4) The recovery rate of the second reference solution is within 95.0% to 105.0%. Taking CA4 as an example, the calculation formula is: Recovery rate % = (peak area of ​​CA4 in STD-2 solution × sample weight of CA4 in STD-1) / (sample weight of CA4 in STD-2 × average peak area of ​​CA4 in the first 5 injections of STD-1 solution). The recovery rate calculation method of BLZ945 and CA4_IM01 is similar and will not be repeated here.

[0025] (5) BLZ945, CA4-IM01, CA4, 2,4,6-trichlorobenzoic acid, CB-PLG-NPs-IM02, and DMAP were respectively prepared into 0.01-0.1 mg / mL solutions as specific solutions to determine the separation degree of the detection method described in the present invention. The acceptance standard of the separation degree is: the separation degree between each peak of BLZ945, CA4, and CA4_IM01 and the adjacent peaks should be ≥1.5.

[0026] The method for detecting small molecules in CB-PLG-NPs drugs provided by the present invention successfully detects small molecules in CB-PLG-NPs drugs, including BLZ945 active small molecules, CA4 active small molecules, and 2,4,6-trichlorobenzoic acid and CA4_IM01, an isomer of CA4, which exist as small molecule impurities, through a gradient elution HPLC detection method. In addition, the content of small molecules in different binding states in CB-PLG-NPs drugs can be detected according to different test sample solutions of the CB-PLG-NPs drugs. Compared with traditional methods, the chromatographic analysis method is fast, accurate, and highly sensitive, and has broad application prospects in industrial production.

[0027] The present invention also provides a method for detecting the drug loading of CB-PLG-NPs drugs, comprising:

[0028] S1) detecting the CB-PLG-NPs drug test solution to obtain the total content and free content of small molecules in the CB-PLG-NPs drug; the detection method is the detection method in the above-mentioned CB-PLG-NPs drug;

[0029] S2) detecting the content of other impurities in the CB-PLG-NPs drug;

[0030] S3) According to the total content and free content of small molecules in the CB-PLG-NPs drug described in step S1) and the content of other impurities in the CB-PLG-NPs drug described in step S2), the drug loading capacity of the CB-PLG-NPs drug is obtained.

[0031] The present invention first detects the total content of small molecules and the free content of small molecules in the CB-PLG-NPs drug test solution, respectively. When the present invention detects the total content of small molecules in the CB-PLG-NPs drug test solution, the preparation method of the CB-PLG-NPs drug test solution is the same as described above and will not be repeated. When the present invention detects the free content of small molecules in the CB-PLG-NPs drug test solution, the preparation method of the CB-PLG-NPs drug test solution is the same as described above and will not be repeated.

[0032] After the total content and free content of small molecules in the CB-PLG-NPs drug test solution are detected, the content of other impurities in the CB-PLG-NPs drug is detected. The other impurities described in the present invention include: water, residual solvents, single-control impurities and residual ions, wherein the residual solvents in the other impurities include: acetone, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, triethylamine, n-heptane, N,N-dimethylformamide, acetic acid, trifluoroacetic acid and 1,2-propylene glycol, and the single-control impurities in the other impurities include mPEG 5K-OH, CB-PLG-NPs_IM02 and DMAP, wherein the CB-PLG-NPs_IM02 is a single-control impurity in the CB-PLG-NPs drug, and its structure is The residual ions in the other impurities include chloride ions and bromide ions. In certain embodiments of the present invention, the water content is detected by Karl Fischer Coulometry, the residual solvent is detected by gas chromatography and IC, the mPEG 5K-OH is detected by HPLC detection method using an evaporative light detector, the CB-PLG-NPs_IM02 is detected by HPLC analysis method, the DMAP is detected by HPLC analysis method, and the residual ions are detected by IC analysis method.

[0033] After detecting the content of other impurities in the CB-PLG-NPs drug, the present invention obtains the drug loading of the CB-PLG-NPs drug according to the total content and free content of small molecules in the CB-PLG-NPs drug and the content of other impurities in the CB-PLG-NPs drug. Specifically, the drug loading of the CB-PLG-NPs drug is the drug loading of CA4 and BLZ945 in the CB-PLG-NPs drug, respectively.

[0034] The content of the CB-PLG-NPs drug of the present invention is %=100%-water%-residual solvent%-mono-control impurities%-residual ions%-free small molecules%. Specifically, the content of the CB-PLG-NPs drug is %=100%-water%-residual solvent%-mono-control impurities (mPEG 5k-OH)%-mono-control impurities (CB-PLG-NPs_IM02)%-mono-control impurities (DMAP)%-residual ions (chloride ions)%-residual ions (bromide ions)%-free active small molecules (BLZ945)%-free active small molecules (CA4)%-free small molecule impurities (CA4_IM01)%-small molecule impurities (2,4,6-trichlorobenzoic acid)%.

[0035] The drug loading amount of CA4 in the CB-PLG-NPs drug of the present invention is = [(CA4 content % - CA4 free content %) / CB-PLG-NPs drug content %] × 100%.

[0036] The drug loading amount of BLZ945 in the CB-PLG-NPs drug of the present invention is = [(BLZ945 content % - BLZ945 free content %) / CB-PLG-NPs drug content %] × 100%.

[0037] The content of CA4_IM01 described in the present invention is %=[(CA4_IM01 peak area in CB-PLG-NPs drug test solution×CA4_IM01 reference substance concentration) / (CA4_IM01 reference substance peak area×sample concentration)]×100%.

[0038] The present invention provides a method for detecting small molecules in CB-PLG-NPs drugs and its application. The method for detecting small molecules provided by the present invention can simultaneously detect the contents of two active small molecules, BLZ945 and CA4, and two small molecule impurities, 2,4,6-trichlorobenzoic acid and CA4_IM01, an isomer of CA4, in CB-PLG-NPs drugs, with high accuracy and sensitivity and strong specificity. The method for detecting the drug loading of CB-PLG-NPs drugs provided by the present invention proposes a complete and systematic set of detection methods, which is conducive to truly reflecting the loading of active substance small molecules of CB-PLG-NPs, can truly compare the amount of its content, can minimize the system error, so as to ensure that accurate quantification is given when administering the drug; and in the whole set of detection methods, all related substances are detected, which can be used as a reference for the promotion of drugs, and as a favorable reference for pharmacology and toxicology. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the synthesis route of CB-PLG-NPs API;

[0040] Figure 2 is the UV spectrum of BLZ945;

[0041] Figure 3 is the UV spectrum of CA4_IM01;

[0042] Figure 4 is the UV spectrum of CA4;

[0043] Figure 5 The HPLC chromatograms of the MIX solution of the present invention observed according to gradient program 1 and gradient program 2 respectively;

[0044] Figure 6 The HPLC chromatogram of the MIX solution of the present invention observed according to the gradient program 3;

[0045] Figure 7 is a schematic diagram of the change of mobile phases A and B over time in the gradient program 3;

[0046] Figure 8 A high performance liquid chromatogram for the sensitivity test of the method of the present invention;

[0047] Fig. 9 A high performance liquid chromatogram for testing the specificity of the method of the present invention;

[0048] Fig.10 It is a high performance liquid chromatogram of residual solvents in the method of the present invention. DETAILED DESCRIPTION

[0049] The present invention discloses a method for detecting small molecules in CB-PLG-NPs drugs and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0050] The present invention will be further described below in conjunction with embodiments:

[0051] Example 1

[0052] Chromatographic conditions and system suitability test:

[0053] (1) Preparation and determination of diluents: A mixed solvent of water and acetonitrile was used as the diluent. This diluent had good solubility (>5 mg / mL) for BLZ945, CA4, CA4_IM01 and 2,4,6-trichlorobenzoic acid. A mixed solvent of water and acetonitrile with a volume ratio of 1:1 was prepared as diluent 1, a mixed solvent of water and acetonitrile with a volume ratio of 2:1 was prepared as diluent 2, and a mixed solvent of water and acetonitrile with a volume ratio of 3:1 was prepared as diluent 3. The solubility of the three diluents in BLZ945, CA4, CA4_IM01 and 2,4,6-trichlorobenzoic acid was tested. The results were all clear and transparent. The concentrations of BLZ945, CA4, CA4_IM01 and 2,4,6-trichlorobenzoic acid could reach above 5 mg / mL. Diluent 1 was selected for subsequent testing.

[0054] (2) Determination of detection wavelength: BLZ945, CA4_IM01 and CA4 were dissolved in acetonitrile solution respectively, and their wavelengths were detected by UV detector, such as Figures 2 to 4 As shown, Figures 2 to 4 They are the UV spectra of BLZ945, CA4_IM01 and CA4, respectively. Figures 2 to 4 It can be observed that at a wavelength of 220 nm, BLZ945, CA4_IM01 and CA4 all have strong absorption, so the detection wavelength is set at 220 nm.

[0055] (3) Determination of gradient program: Prepare MIX solution (containing CA4, BLZ945, CA4_IM01, 2,4,6-trichlorobenzoic acid at a concentration of about 0.05 mg / mL, acetonitrile: water volume ratio = 1:1), and examine its spectrum according to the three gradient programs shown in Table 1:

[0056] Table 1

[0057]

[0058] The results of the investigation are as follows Figure 5 and Figure 6 As shown, Figure 5 1 and 2 are high performance liquid chromatograms of the MIX solution of the present invention respectively observed according to gradient program 1 and gradient program 2. Figure 6 The HPLC chromatogram of the MIX solution of the present invention according to the gradient program 3. Figure 5 It can be seen that both gradient program 1 and gradient program 2 have a peak, which interferes with the detection to a certain extent. Figure 6 No peak is visible, so select gradient program 3, such as Figure 7 As shown, Figure 7 Schematic diagram of the change of mobile phase A and B over time in the gradient program 3.

[0059] (4) Method sensitivity test

[0060] ① Preparation of reference solution: 25±2.5 mg of each of reference substances BLZ945, CA4 and CA4_IM01 were accurately weighed and placed in a 50 mL volumetric flask, and the diluent was added to dissolve by ultrasonic (room temperature, 10 s), and allowed to stand at room temperature, and the diluent was continued to be added to the scale, and the mixture was shaken and marked as STD-Stock-1. Accurately transfer 1 mL of STD-Stock-1 to a 10 mL volumetric flask, and the diluent was added to the scale, and the mixture was shaken and marked as STD-1 as the reference solution.

[0061] ② Preparation of sensitivity solution: Prepare sensitivity solution (BLZ945, CA4, CA4_IM01 concentrations are <0.0005 mg / mL respectively): Accurately pipette 1 mL of the above STD-1 solution into a 200 mL volumetric flask, add diluent to the scale, shake well, and mark as LOQ.

[0062] ③Sensitivity test: Select gradient program 3 to test LOQ. The results are as follows: Figure 8 As shown in Table 2, Figure 8 This is a high performance liquid chromatogram of the sensitivity test of the method of the present invention:

[0063] Table 2

[0064] sample Sensitivity (S / N) standard BLZ945 44 ≥10 CA4 20 ≥10 CA4_IM01 28 ≥10

[0065] From the results in Table 2, it can be seen that the sensitivity of gradient program 3 to BLZ945, CA4 and CA4_IM01 all meet the requirements.

[0066] (5) Specificity test: Prepare the solutions shown in Table 3, select gradient program 3 and perform HPLC on each solution. The results are as follows: Fig. 9 As shown, Fig. 9 The specificity test of the method of the present invention is shown in the following high performance liquid chromatogram:

[0067] Table 3

[0068]

[0069] from Figure 8 It can be seen that under the chromatographic conditions, other known impurities 2,4,6-trichlorobenzoic acid, CB-PLG-NPS_IM02 and DMAP had no interference at the CA4, BLZ945 and CA4_IM01 peaks, indicating that the analytical method has good specificity.

[0070] (6) Effects of flow rate and column temperature on detection methods

[0071] ① Preparation of test solution: Take 20±2 mg of CB-PLG-NPs sample, accurately weigh it, place it in a 50 mL volumetric flask, add 20 mL of H 2 O and 1 mL of 1 mol / L NaOH solution, and sonicate at room temperature in the dark for 2 h. Then, add 20 mL of acetonitrile and sonicate for 1 h in the dark. Cool to room temperature, add diluent to the volume to the mark, and mark it as SPL-1-1 as the test solution.

[0072] ② Detection of drug loading of the test solution at different flow rates: Select gradient program 3, and detect the contents of BLZ945, CA4 and CA4_IM01 in SPL-1-1 at flow rates of 1.4 mL / min, 1.5 mL / min and 1.6 mL / min, respectively. The results are shown in Table 4:

[0073] Table 4

[0074]

[0075] It can be seen from Table 4 that different flow rates do not affect the test results.

[0076] ③ Detection of drug loading of the test solution at different column temperatures: Select gradient program 3, and detect the contents of BLZ945, CA4 and CA4_IM01 in SPL-1-1 at column temperatures of 38°C, 40°C and 42°C, respectively. The results are shown in Table 5:

[0077] Table 5

[0078]

[0079] It can be seen from Table 5 that different column temperatures do not affect the test results.

[0080] Example 2

[0081] S1) Detection of total drug content:

[0082] S1-1) Preparation of diluent

[0083] A mixed solvent of acetonitrile and water was used as the diluent, and the volume ratio of water:acetonitrile was selected to be 1:1.

[0084] S1-2) Preparation of reference solution

[0085] Weigh 25±2.5 mg of each of the reference substances BLZ945, CA4 and CA4_IM01 accurately, place in a 50 mL volumetric flask, add diluent and sonicate at room temperature for 10 seconds to dissolve, place at room temperature, add diluent to the mark, shake well, and mark as STD-Stock-1; accurately transfer 1 mL of STD-Stock-1 to a 10 mL volumetric flask, add diluent to the mark, shake well, and mark as STD-1 as the reference solution. Prepare STD-2 in the same way as a calibration solution.

[0086] S1-3) Preparation of sensitivity solutions (BLZ945, CA4 and CA4_IM01 concentrations < 0.0005 mg / mL respectively)

[0087] Accurately pipette 1 mL of STD-1 solution into a 200 mL volumetric flask, add diluent to the mark, shake well, and mark as LOQ.

[0088] S1-4) Preparation of test solution

[0089] The CB-PLG-NPs samples with batch numbers P09442-042-P3 and P09442-046-P1 were prepared into test solutions respectively. The specific steps were as follows: 25±2.5 mg of CB-PLG-NPs sample was accurately weighed and placed in a 50 mL volumetric flask. 20 mL of H 2 O and 1mL of 1mol / L NaOH solution were mixed, and the mixture was sonicated at room temperature in the dark for 2 hours. Then 20mL of acetonitrile was added to the mixture in the dark for 1 hour. The mixture was cooled to room temperature, and the diluent was added to the volume to the mark. The mixture was marked as SPL-1-1 as the test solution. SPL-1-2 of the same batch was prepared in the same way as a parallel sample.

[0090] S1-5) Preparation of blank solution

[0091] Take a 50mL volumetric flask, add 25mL of pure water and 1mL of 1mol / L NaOH solution, mix, then add 20mL of acetonitrile and cool to room temperature, add diluent to make up the volume, shake well, and use this as the blank solution, marked as Blank.

[0092] S1-6) High performance liquid chromatography detection

[0093] The blank solution, the sensitive solution, the reference solution, and the test solution were detected by high performance liquid chromatography according to the gradient program 3 described in Example 1 to obtain the peak areas, and the drug content in the sample was calculated. The number of injection needles is shown in Table 6:

[0094] Table 6

[0095] Serial Number Sample name Number of injection needles 1 Blank solution 2～5 2 Sensitivity solution (LOQ) 1 3 Reference solution (STD-1) 5 4 Reference solution (STD-2) 1 5 Test solution (SPL-1-2) 1 6 Test solution (SPL-1-2) 1

[0096] The above HPLC tests complied with the following system usability acceptance criteria:

[0097] ① The blank solution has no interference at the peak positions of CA4, BLZ945, and CA4_IM01. If there is interference, the peak area of ​​the interference peak shall not be greater than the peak area of ​​each component in the sensitivity solution (≤LOQ), otherwise it should be evaluated.

[0098] ②The signal-to-noise ratio of CA4_IM01 in the sensitivity solution (LOQ) should be ≥10.

[0099] ③ The system precision requires that the RSD of the peak area of ​​each component in the STD-1 chromatogram of 5 consecutive injections is ≤5.0%, and the RSD of the retention time is ≤1.0%.

[0100] ④ The recovery rate of STD-2 reference substance is within 95.0% to 105.0%. Taking CA4 as an example, the recovery rate % = peak area of ​​CA4 in STD-2 solution × sample weight of CA4 in STD-1 / (sample weight of CA4 in STD-2 × average peak area of ​​CA4 in the first 5 injections of STD-1 solution).

[0101] ⑤ Preparation of specific solutions: BLZ945, CA4_IM01, CA4 and 2,4,6-trichlorobenzoic acid were prepared into 0.005 mg / mL solution, CB-PLG-NPs-IM02 was prepared into 0.01 mg / mL solution, and DMAP was prepared into 0.5 mg / mL solution. The acceptance standard of separation degree is: the separation degree of each peak of BLZ945, CA4_IM01, CA4 and the adjacent peak should be ≥1.5.

[0102] The total drug content was calculated according to the peak area obtained by HPLC of the test solution and the peak area of ​​the reference solution. The results are shown in Table 7:

[0103] Table 7

[0104]

[0105] S2) Detection of free drug content

[0106] S2-1) Preparation of each solution: prepare diluent, blank solution and sensitivity solution according to the method described in S1);

[0107] S2-2) Preparation of reference solution: Take 25±2.5 mg of BLZ945 reference, CA4 reference, CA4_IM01 reference and 2,4,6-trichlorobenzoic acid reference respectively, weigh accurately, place in a 50 mL volumetric flask, add diluent to dissolve by ultrasonic, place at room temperature, add diluent to scale, shake well, and mark as STD-Stock-1; accurately transfer 1 mL of STD-Stock-1 to a 100 mL volumetric flask, add diluent to scale, shake well, and mark as STD-1. Prepare STD-2 in the same way for calibration.

[0108] S2-3) Preparation of test solution: CB-PLG-NPs samples with batch numbers P09442-042-P3 and P09442-046-P1 were prepared into test solution by accurately weighing 25±2.5 mg of sample and placing it in a 50 mL volumetric flask, adding 10 mL of ACN to fully dissolve the sample, and adding about 35 mL of H 2 O, shake well, leave at room temperature for 1 hour, then add water to make up to volume to obtain the test solution.

[0109] S2-4) HPLC detection: The two batches of test sample solutions prepared in S2-3) were subjected to HPLC detection according to the gradient program 3 described in Example 1.

[0110] The step of calculating the drug residue content according to the peak area obtained by the HPLC detection of the test solution and the peak area of ​​the reference substance, the results are shown in Table 8:

[0111] Table 8

[0112]

[0113] S3) Determine the content of CB-PLG-NPs

[0114] S3-1) Determination of moisture content in CB-PLG-NPs;

[0115] The Karl Fischer Coulometry method was used to determine the CB-PLG-NPs samples with batch numbers P09442-042-P3 and P09442-046-P1, respectively. The specific steps are as follows: accurately weigh two samples of 100 mg each, place the samples in the sample tray, set the instrument parameters, wait for 60 seconds, stop point at 250.0 mV, titer of 5, and the reagent is methanol. After the titration is completed, record the measurement data, and take the average of the two results. The results are shown in Table 9:

[0116] Table 9

[0117]

[0118] S3-2) Determination of some residual solvent contents in CB-PLG-NPs;

[0119] Gas chromatography was used to detect some residual solvents in CB-PLG-NPs, including acetone, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, triethylamine, n-heptane and N,N-dimethylformamide. A Shimadzu GC-2030 gas chromatograph (with FID detector) or equivalent instrument was used, the diluent was 5% ammonia in N-methylpyrrolidone, and the blank solvent was the same. Chromatographic conditions: carrier gas was N 2 , injection port temperature 240℃, split ratio 10:1, detector temperature 260℃, injection volume 1μL, hydrogen flow rate 32mL / min, air flow rate 200mL / min, detection time 30min, program temperature rise 10℃ / min, from 40℃ (stay 5min) to 240℃ (stay 5min).

[0120] Preparation of reference solution: Take 500 mg each of acetone, methyl tert-butyl ether, ethyl acetate, triethylamine, and n-heptane, 72 mg of tetrahydrofuran, and 88 mg of N,N-dimethylformamide, weigh accurately, and place in a 50 mL volumetric flask with a small amount of the above diluent, dissolve and dilute to the mark with the above diluent, shake well, and mark as Stock-1. Take 1 mL of the mixed solvent and place in a 100 mL volumetric flask with a small amount of the above diluent, dilute to the mark with the above diluent, shake well, and mark as STD-1. Prepare STD-2 in the same way.

[0121] Preparation of sample solution: Take about 40 mg of CB-PLG-NPs samples with batch numbers P09442-042-P3 and P09442-046-P1, accurately weigh them, place them in a 2 mL volumetric flask, add the above-mentioned diluent to dissolve them, and then dilute to the scale. The obtained sample solutions are marked as SPL-1 and SPL-2.

[0122] Calculation of residual solvent content (w / w) = ASPL × WSTD × VSPL / (ASTD × VSTD × WSPL) × 100%, where ASPL refers to the peak area of ​​each target solvent in the sample solution; WSPL refers to the sample weight (mg); VSPL refers to the volume of the sample solution (mL); ASTD refers to the average peak area of ​​each target solvent in 6 consecutive injections of STD-1; WSTD refers to the sample weight (mg) of each target solvent in STD-1; VSTD refers to the total dilution volume (mL) of each target solvent in STD-1. The detection results of each residual solvent are as follows: Fig.10 As shown in Table 10, Fig.10 The residual solvent high performance liquid chromatogram of the method of the present invention is:

[0123] Table 10

[0124]

[0125] S3-3) Determination of the residual solvent 1,2-propylene glycol content in CB-PLG-NPs;

[0126] The detection was performed using Shimadzu GC-2030 gas chromatograph (with FID detector) or equivalent instrument, the diluent was acetonitrile, and the blank solvent was the same as the diluent. Gas chromatography conditions: DB-WAX (30m×0.25mm, 0.25μm) chromatographic column, FID detector, N 2 The carrier gas flow rate was 1.2 mL / min, the injection port temperature was 240°C, the split ratio was 30 / 1, the FID detector temperature was 260°C, the running time was 30.5 min, the temperature was programmed at 10°C / min, and the temperature was increased from 40°C (stayed for 5 min) to 245°C (stayed for 5 min).

[0127] Preparation of reference solution: Take about 50 mg of 1,2-propylene glycol, weigh accurately, and place it in a 20 mL volumetric flask containing a small amount of the above diluent. Dissolve it with the above diluent and dilute it to the mark. Shake well. Take 1 mL of the solution and dilute it with the above diluent to a 100 mL volumetric flask. Mark it as STD-4-1. Prepare STD-4-2 in the same way.

[0128] Preparation of sample solution: Take about 25 mg of CB-PLG-NPs samples with batch numbers P09442-042-P3 and P09442-046-P1, accurately weigh them, place them in a 5 mL volumetric flask, add the above-mentioned diluent to dissolve, and then dilute to the scale. The obtained sample solutions are marked as SPL-1 and SPL-2.

[0129] Calculation of residual content of 1,2-propylene glycol (w / w) = [(ASPL×WSTD×VSPL) / (ASTD×VSTD×WSPL)]×100%; where ASPL refers to the peak area of ​​1,2-propylene glycol in the sample solution; WSPL refers to the sample weight of 1,2-propylene glycol (mg); VSPL refers to the volume of 1,2-propylene glycol solution (mL); ASTD refers to the average value of the peak area of ​​1,2-propylene glycol in 6 consecutive injections of STD-1; WSTD refers to the sample weight of 1,2-propylene glycol in STD-1 (mg); VSTD refers to the total dilution volume of 1,2-propylene glycol in STD-1 (mL). The test results are shown in Table 11:

[0130] Table 11

[0131] batch number 1,2-Propanediol P09442-042-P3 <500ppm P09442-046-P1 <500ppm

[0132] S3-4) Determination of the residual content of the single-control impurity mPEG 5K-OH in CB-PLG-NPs;

[0133] The HPLC detection method using an evaporative light detector was used, and the chromatographic parameters were as follows: chromatographic column ChromCore SAA 150×4.6mm, 5μm or equivalent column, column temperature 35°C, flow rate 1.0mL / min, evaporative light detector, evaporation temperature 60°C, injection volume 10μL. Chromatographic procedure: gradient elution, mobile phase A was 0.1% HAc in H 2 O solution (V / V), mobile phase B was isopropanol, and mobile phase B was changed from 5% to 80% in 15 min;

[0134] The diluent is a mixed solution of acetonitrile and water in a volume ratio of 1:1;

[0135] Preparation of reference solution: mPEG 5k-OH was prepared in the following order: 0.6 mg / mL, 0.3 mg / mL, 0.15 mg / mL, 0.06 mg / mL and 0.015 mg / mL;

[0136] The sensitivity solution is 0.015 mg / mL reference solution

[0137] Sample solution preparation: 150 mg of CB-PLG-NPs samples of batch numbers P09442-042-P3 and P09442-046-P1 were respectively dissolved in 2 mL of DMF and diluted to 5 mL with the above diluent. The obtained sample solutions were marked as SPL-1 and SPL-2.

[0138] Preparation of standard curve: Draw the standard curve with the log value of the reference substance concentration as the horizontal axis and the log value of the corresponding peak area as the vertical axis; calculate the content of mPEG 5k-OH using the external standard method, and the results are shown in Table 12.

[0139] Table 12

[0140] batch number mPEG 5k-OH P09442-042-P3 2.7％ P09442-046-P1 0.8％

[0141] S3-5) Determination of the content of single controlled impurity CB-PLG-NPs_IM02 in CB-PLG-NPs

[0142] The HPLC analysis method is adopted, and the chromatogram is as follows: Shimadzu LC-20A liquid chromatograph with PDA detector or equivalent instrument is used, the chromatographic column is a waters phenylhexyl column or equivalent chromatographic column, the column temperature is 40°C, and the detection wavelength is 220nm. Chromatographic procedure: mobile phase A is 0.03% TFA aqueous solution, B is 0.03% TFA acetonitrile solution; isocratic method 15min mobile phase B is 65%.

[0143] Preparation of reference solution: Take about 25 mg of CB-PLG-NPs_IM02 reference substance and place it in a 50 mL volumetric flask, add 3 mL of THF to dissolve it, then add acetonitrile to make up the volume, transfer 1 mL to a 100 mL volumetric flask, add acetonitrile to make up the volume and shake well, mark it as STD-1, and prepare STD-2 in the same way.

[0144] Preparation of sensitivity solution: Pipette 1 mol / L STD-1 solution into a 20 mL volumetric flask, add acetonitrile to dilute to the mark, shake well, and mark as LOQ.

[0145] Sample solution preparation: Take 25 mg of CB-PLG-NPs samples with batch numbers P09442-042-P3 and P09442-046-P1, accurately weigh them, place them in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, and the resulting sample solutions are marked as SPL-1 and SPL-2. The content of CB-PLG-NPs_IM02 was calculated using the external standard method, and the results are shown in Table 13.

[0146] Table 13

[0147] batch number CB-PLG-NPs_IM02 P09442-042-P3 ＜0.05％ P09442-046-P1 ＜0.05％

[0148] S3-6) Determination of the content of single-control impurity DMAP in CB-PLG-NPs

[0149] The HPLC analysis method was used. Chromatographic conditions: Shimadzu LC-20A series liquid chromatograph with PDA detector or equivalent instrument was used. The chromatographic column was Waters Xselect column or equivalent column. The column temperature was 35°C. The detection wavelength was 280nm. The chromatographic procedure was gradient elution procedure. Mobile phase A was 0.03% HFA. 2 O solution, mobile phase B was acetonitrile, mobile phase B changed from 5% to 90% in 15 min, and the overall acquisition time was 22 min.

[0150] Preparation of reference substance: Accurately weigh 25 mg of DMAP and diluent (a mixed solution of acetonitrile and water with a volume ratio of 1:1) and dilute it into a 100 mL volumetric flask, shake well, then transfer 1 mL to a 100 mL volumetric flask, add the above diluent to the scale, shake well, mark it as STD-1, and prepare STD-2 in the same way.

[0151] The sensitivity solution was 0.00015 mg / mL of DMAP.

[0152] Sample solution: Accurately weigh about 25 mg of CB-PLG-NPs samples with batch numbers P09442-042-P3 and P09442-046-P1 in a 50 mL volumetric flask, add the above diluent to dissolve by ultrasonication, place at room temperature, add the above diluent to dilute to the scale, shake well, mark as SPL-1, and prepare SPL-2 in the same way. The DMAP content was calculated by the external standard method, and the results are shown in Table 14.

[0153] Table 14

[0154] batch number DMAP P09442-042-P3 ＜0.03％ P09442-046-P1 0.03％

[0155] S3-7) Determination of residual solvent acetic acid content in CB-PLG-NPs

[0156] IC analysis method was used, using Thermo ICS-5000+ ion chromatograph or equivalent instrument; ThermoDionex IonPac AS11-HC (4mm×250mm) or equivalent ion chromatography column was used. Column temperature was 30℃, detection cell temperature was 35℃, injection volume was 25μL; suppression current was 87mA, and the detector was a conductivity detector. Chromatographic procedure: KOH concentration was 1mmol / L within 15min; flow rate was 1mL / min;

[0157] The diluent was 5% MeOH aqueous solution (V / V), and the blank solvent was the same.

[0158] Preparation of reference solution: accurately transfer 2 mL of acetate ion standard solution into a 10 mL volumetric flask, dilute to the mark with ultrapure water, shake well, accurately transfer 1 mL into a 20 mL volumetric flask, add 1 mL of MeOH and dilute to the mark with ultrapure water, shake well, mark as STD-1, and prepare STD-2 in the same way.

[0159] The sensitivity solution is 0.003 mg / mL acetic acid

[0160] Sample solution preparation: Take about 40 mg of CB-PLG-NPs samples with batch numbers P09442-042-P3 and P09442-046-P1, accurately weigh, place in a 20 mL volumetric flask, add 1 mL of MeOH to completely dissolve the sample, then add ultrapure water to dilute to the scale, shake well, mark as SPL-1, and prepare SPL-2 in the same way. The content of acetic acid in CB-PLG-NPs was calculated using the external standard method, and the results are shown in Table 15:

[0161] Table 15

[0162] batch number Acetic acid P09442-042-P3 <500ppm P09442-046-P1 <500ppm

[0163] S3-8) Determination of residual solvent (trifluoroacetic acid) and residual ion (chloride and bromide) contents in CB-PLG-NPs;

[0164] IC analysis method was used, using Thermmo ICS-5000+ ion chromatograph or equivalent instrument, Dionex IonPac AS19 (4mm×50mm) chromatographic column, flow rate was 1mL / min, column temperature was 30℃, detection cell temperature was 35℃, injection volume was 25μL, suppressor current was 87mA, and the detector was a conductivity detector. Chromatographic procedure: KOH concentration was 15mmol / L within 15min.

[0165] The diluent was 5% MeOH aqueous solution (V / V), and the blank solvent was the same.

[0166] Preparation of reference solution: Weigh 50 mg of trifluoroacetic acid reference substance, accurately weigh and place in a 25 mL volumetric flask, add ultrapure water to dissolve and dilute to the scale, accurately transfer 1 mL of trifluoroacetic acid solution and 0.6 mL of bromide ion standard solution into a 10 mL volumetric flask, add ultrapure water to dilute to the scale, shake well, accurately transfer 1 mL of trifluoroacetic acid bromide ion mixed solution and 0.8 mL of chloride ion standard solution into a 20 mL volumetric flask, add 1 mL of MeOH, and then dilute to the scale with ultrapure water, shake well, mark as STD-1, and prepare STD-2 solution in the same way.

[0167] Sensitivity solution (containing 0.001 mg / mL trifluoroacetic acid, 0.0003 mg / mL bromide ions, and 0.004 mg / mL chloride ions).

[0168] Sample solution preparation: Take about 40 mg of CB-PLG-NPs sample, accurately weigh it, place it in a 20 mL volumetric flask, add 1 mL of MeOH to completely dissolve the sample, then add ultrapure water to dilute to the scale and shake well. The contents of trifluoroacetic acid, chloride ions and bromide ions in CB-PLG-NPs were calculated using the external standard method, and the results are shown in Table 16.

[0169] Table 16

[0170] batch number Trifluoroacetic acid Chloride ion Bromide ion P09442-042-P3 4107ppm 1.00％ ＜0.02％ P09442-046-P1 <150ppm 1.08％ ＜0.02％

[0171] S3-9) Obtaining the CB-PLG-NPs content and drug loading of the two batches of drugs according to the above-determined content;

[0172] CB-PLG-NPs drug content % = 100% - moisture % - residual solvent % - single controlled impurity % - residual ion % - free small molecule % = 100% - moisture % - residual solvent % - single controlled impurity (mPEG 5k-OH) % - single controlled impurity (CB-PLG-NPs_IM02) % - single controlled impurity (DMAP) % - residual ion (chloride ion) % - residual ion (bromide ion) % - free active small molecule (BLZ945) % - free active small molecule (CA4) % - free small molecule impurity (CA4_IM01) % - small molecule impurity (2,4,6-trichlorobenzoic acid) %;

[0173] Drug loading (CA4) = (CA4 content% - CA4 free content%) / CB-PLG-NPs drug content% = [(CA4 content% - CA4 free content%) / (100% - water% - residual solvent% - single-control impurities% - residual ions% - free small molecules%)] × 100%;

[0174] Drug loading (BLZ945) = (BLZ945 content% - BLZ945 free content%) / CB-PLG-NPs drug content% = [(BLZ945 content% - BLZ945 free content%) / (100% - water% - residual solvent% - single-control impurities% - residual ions% - free small molecules%)] × 100%;

[0175] CA4_IM01 content% = CA4_IM01 peak area in CB-PLG-NPs drug test solution × CA4_IM01 reference concentration / CA4_IM01 reference peak area / sample concentration × 100%. (Used for drug quality control)

[0176] The results are shown in Table 17:

[0177] Table 17

[0178]

[0179]

[0180] Note: Values ​​below LOQ are calculated as 0.

[0181] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting small molecules in CB-PLG-NPs drugs, It is characterized in that include: HPLC was used to detect the CB-PLG-NPs drug test solution; The chromatographic conditions of the HPLC are: A 0.01-0.1 wt% trifluoroacetic acid aqueous solution was used as mobile phase A, a 0.01-0.1 wt% acetonitrile aqueous solution was used as mobile phase B, and gradient elution was performed. The procedure of the gradient elution was as follows: 。 2. The detection method according to claim 1, It is characterized in that The detection wavelength of the HPLC is 205nm-230nm.

3. The detection method according to claim 1, It is characterized in that The HPLC column is a reverse phase C 18 Chromatographic column.

4. The detection method according to claim 1, It is characterized in that The column temperature of the HPLC chromatographic column is 20°C to 50°C.

5. The detection method according to claim 1, It is characterized in that The flow rates of the mobile phase A and the mobile phase B are 0.5 mL / min to 1.6 mL / min.

6. The detection method according to claim 1, It is characterized in that The preparation method of the CB-PLG-NPs drug test solution comprises: Water, NaOH and acetonitrile were used as extraction solvents to prepare CB-PLG-NPs drug test solution; or, The CB-PLG-NPs drug test solution was prepared using acetonitrile aqueous solution as the extraction solvent.

7. A method for detecting the drug loading of CB-PLG-NPs drugs, It is characterized in that include: S1) detecting the CB-PLG-NPs drug test solution to obtain the total content and free content of small molecules in the CB-PLG-NPs drug; the detection method is the detection method described in any one of claims 1 to 6; S2) detecting the content of other impurities in the CB-PLG-NPs drug; S3) According to the total content and free content of small molecules in the CB-PLG-NPs drug described in step S1) and the content of other impurities in the CB-PLG-NPs drug described in step S2), the drug loading capacity of the CB-PLG-NPs drug is obtained.

8. The drug loading detection method according to claim 7, It is characterized in that In step S1), when the total content of small molecules in the CB-PLG-NPs drug test solution is detected, the preparation method of the CB-PLG-NPs drug test solution includes: using water, NaOH and acetonitrile as extraction solvents to prepare the CB-PLG-NPs drug test solution.

9. The drug loading detection method according to claim 7, It is characterized in that In step S1), when the free content of small molecules in the CB-PLG-NPs drug test solution is detected, the preparation method of the CB-PLG-NPs drug test solution includes: using acetonitrile aqueous solution as an extraction solvent to prepare the CB-PLG-NPs drug test solution.

10. The drug loading detection method according to claim 7, It is characterized in that In step S2), the other impurities include: water, residual solvent, single-control impurities and residual ions; The residual solvent includes: acetone, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, triethylamine, n-heptane, N,N-dimethylformamide, acetic acid, trifluoroacetic acid and 1,2-propylene glycol; The single controlled impurities include mPEG 5K-OH, CB-PLG-NPs_IM02 and DMAP; The residual ions include chloride ions and bromide ions.

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