Method for detecting content of a lobaplatin intermediate

Abstract

The present application belongs to the technical field of pharmaceutical analysis and quality control, and relates to a detection method for the content of a lobaplatin intermediate, in particular to a detection method for the content of trans-1,2-diaminocyclobutane diiodoplatinum (II), which is an HPLC method, and the detection conditions are as follows: octadecyl bonded silica is used as a filler, an ammonium acetate solution is used as a mobile phase A, and a mixed solution of the ammonium acetate solution and acetonitrile is used as a mobile phase B, and gradient elution is performed. The detection method has strong specificity, good linear relationship, high accuracy and high precision. The present application has important guiding significance for the quantity of material feeding in subsequent reactions, the improvement of the yield and purity of lobaplatin by establishing the detection method for the content of the lobaplatin intermediate trans-1,2-diaminocyclobutane diiodoplatinum.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis and quality control technology, and relates to a method for detecting the content of lobaplatin intermediates, specifically a method for detecting the content of trans-1,2-diaminocyclobutane diiodoplatin(II). Background Technology

[0002] Lobaplatin (D19466), also known as lobaplatin, is a third-generation platinum-based antitumor drug following cisplatin and carboplatin. Its chemical name is cis-[trans-1,2-cyclobutanebis(methylamine)-N,N']-[(2S)-lactic acid-O1,O2]-platinum(II), and its molecular formula is C9H. 18 N₂O₃Pt, with a molecular weight of 397.34, has the following chemical structural formula (a):

[0003]

[0004] Lobaplatin possesses alkylating activity and belongs to the alkylating agent category (in a broad sense). It exhibits good antitumor activity, such as strong inhibitory effects on isolated AH135-tumor, B16-melanoma, and colon cancer 115, as well as in vivo mouse P338 leukemia. Lobaplatin is characterized by its strong anticancer activity, low toxicity, lack of cumulative toxicity and nephrotoxicity, and relatively low bone marrow toxicity. Currently marketed injectable lobaplatin is mainly used for the treatment of breast cancer, small cell lung cancer, and chronic myeloid leukemia. Among these, trans-1,2-diaminocyclobutane diiodoplatin(II) is an important intermediate in the synthesis of lobaplatin. Accurate determination of its content is crucial for guiding the feeding quantities of subsequent reaction materials and improving the yield and purity of lobaplatin. Summary of the Invention

[0005] The technical problem solved by this invention is that there is no reported method in the prior art for detecting the content of trans-1,2-diaminocyclobutane diiodoplatin(II), an intermediate of lobaplatin. To address this problem, this invention provides a high-precision method for detecting the content of trans-1,2-diaminocyclobutane diiodoplatin(II), which is beneficial for quality control in the lobaplatin preparation process.

[0006] Specifically, the present invention is achieved through the following technical solution:

[0007] This invention provides a method for detecting the content of lobaplatin intermediates, wherein the lobaplatin intermediate is trans-1,2-diaminocyclobutane diiodoplatin(II), and its structural formula is:

[0008]

[0009] The detection method is HPLC, and the detection conditions of HPLC are as follows: using octadecylsilane-bonded silica gel as the packing material, using ammonium acetate solution as mobile phase A, and a mixed solution of ammonium acetate solution and acetonitrile as mobile phase B, and performing gradient elution.

[0010] In some embodiments, the concentration of ammonium acetate solution in mobile phase A is 8–12 mmol / L.

[0011] In some embodiments, the concentration of ammonium acetate solution in mobile phase A is 9–11 mmol / L.

[0012] In some embodiments, the volume ratio of ammonium acetate solution to acetonitrile in the mobile phase B is 1:(5-19).

[0013] In some embodiments, the volume ratio of ammonium acetate solution to acetonitrile in the mobile phase B is 1:(5-15).

[0014] In some embodiments, the concentration of ammonium acetate solution in mobile phase B is 8–12 mmol / L.

[0015] In some embodiments, the concentration of ammonium acetate solution in mobile phase B is 9–11 mmol / L.

[0016] In some implementations, the method for detecting the content of lobaplatin intermediates includes the following steps:

[0017] (1) Preparation of reference solution

[0018] Weigh out trans-1,2-diaminocyclobutane diiodoplatin reference standard and disperse it in N,N-dimethylformamide;

[0019] (2) Preparation of the test solution

[0020] Weigh out the trans-1,2-diaminocyclobutane diiodoplatinum test sample and disperse it in N,N-dimethylformamide;

[0021] (3) High performance liquid chromatography analysis

[0022] The gradient elution method for the high-performance liquid chromatography analysis is as follows:

[0023] 0–25 minutes: Mobile phase A decreases from 100% to 10% by volume, and mobile phase B increases from 0% to 90% by volume.

[0024] In some implementations, the gradient elution time is increased by 2 to 3 minutes in the gradient elution method.

[0025] In some implementations, the gradient elution time is reduced by 2 to 3 minutes in the gradient elution method.

[0026] In some implementations, the flow rate in the HPLC detection conditions is 0.8–1.2 mL / min.

[0027] In some implementations, the column temperature in the HPLC detection conditions is 30–45°C.

[0028] In some implementations, the detection wavelength in the HPLC method is 219–221 nm.

[0029] In some implementations, the resolution between the main peak of compound (II) and each analyte in the chromatogram of the system suitability test solution is not less than 1.5.

[0030] In some implementations, the method for detecting the content of the lobaplatin intermediate trans-1,2-diaminocyclobutane diiodoplatin(II) is used for the quality control of the lobaplatin intermediate raw material or preparation.

[0031] The beneficial effects achieved by this invention are as follows:

[0032] This invention provides a more precise HPLC method for detecting the content of trans-1,2-diaminocyclobutane diiodoplatin(II). This detection method has high specificity, high accuracy, high precision, and good repeatability. The accurate determination of the content of trans-1,2-diaminocyclobutane diiodoplatin(II) in this invention is beneficial for the quality control of the lobaplatin preparation process and has important guiding significance for the amount of materials fed into subsequent reactions and for improving the yield and purity of lobaplatin. Attached Figure Description

[0033] Figure 1 This is the liquid chromatogram of the test solution determined in Example 2-2.

[0034] Figure 2 This is a liquid chromatogram of the test solution determined in Examples 2-3.

[0035] Figure 3 This is a liquid chromatogram of the test solution determined in Examples 2-4.

[0036] Figure 4 This is the liquid chromatogram of the reference standard STD-1 solution in the specificity test of Example 3.

[0037] Figure 5 This is the liquid chromatogram of the reference standard STD-2 solution in the specificity test of Example 3.

[0038] Figure 6 This is the liquid chromatogram of the reference standard STD-3 solution in the specificity test of Example 3.

[0039] Figure 7This is the liquid chromatogram of the SPL solution used in the specificity test of Example 3.

[0040] Figure 8 This is the liquid chromatogram of the test solution in the solution stability test of Example 3.

[0041] Figure 9 This is the liquid chromatogram of the reference solution in the solution stability test of Example 3.

[0042] Figure 10 This is a line graph of diiodide in the linear range test of Example 3.

[0043] Figure 11 This is the liquid chromatogram of the linear solution in the linear range test of Example 3.

[0044] Figure 12 This is the liquid chromatogram of the test solution in the precision test of Example 3.

[0045] Figure 13 is a liquid chromatogram of the reference standard STD-1 solution in the precision test of Example 3.

[0046] Figure 14 This is the liquid chromatogram of the reference standard STD-2 solution in the precision test of Example 3.

[0047] Figure 15 This is the liquid chromatogram of the reference standard STD-bracket solution in the precision test of Example 3.

[0048] Figure 16 is a liquid chromatogram of the intermediate precision solution in the precision test of Example 3. Detailed Implementation

[0049] The purpose of this invention is to provide a method for detecting the content of lobaplatin intermediates, specifically involving an HPLC method for detecting the content of trans-1,2-diaminocyclobutane diiodoplatin(II).

[0050] In one specific embodiment of the present invention, a method for detecting lobaplatin intermediates is provided, wherein the lobaplatin intermediate is trans-1,2-diaminocyclobutane diiodoplatin(II) (also known as diiodide) with the following structure:

[0051]

[0052] The reaction route for preparing lobaplatin from the lobaplatin intermediate trans-1,2-diaminocyclobutane-diiodoplatin(II) described in this invention is as follows:

[0053]

[0054] The detection method is HPLC, and the specific detection conditions for HPLC are as follows:

[0055] Octadecylsilane-bonded silica gel was used as the packing material; mobile phase A consisted of an 8–12 mmol / L ammonium acetate solution, and mobile phase B consisted of a mixture of an 8–12 mmol / L ammonium acetate solution (using water as a solvent) and acetonitrile. The volume ratio of ammonium acetate solution to acetonitrile in mobile phase B was 1:(5–19), and the concentration of ammonium acetate solution was 8–12 mmol / L. The flow rate was 0.8–1.2 mL / min; the column temperature was 30–45 °C; the detection wavelength was 219–221 nm; and gradient elution was performed.

[0056] The gradient elution method is as follows:

[0057] 0–25 minutes: Mobile phase A decreases from 100% to 10% by volume, and mobile phase B increases from 0% to 90% by volume.

[0058] In another specific embodiment of the present invention, the gradient elution time range is increased by 2 to 3 minutes. For example, the gradient elution time is increased by 2 minutes: 0 to 27 minutes: mobile phase A is reduced from 100% to 10% by volume, and mobile phase B is increased from 0% to 90% by volume.

[0059] In another specific embodiment of the present invention, the gradient elution time range is reduced by 2 to 3 minutes in the gradient elution method. For example, the gradient elution time is reduced by 2 minutes: 0 to 23 minutes: mobile phase A is reduced from 100% by volume to 10% by volume, and mobile phase B is increased from 0% by volume to 90% by volume.

[0060] In a preferred embodiment, the test solution is in N,N-dimethylformamide as a solvent; and / or, the reference solution is in N,N-dimethylformamide as a solvent.

[0061] In a preferred embodiment, in the chromatogram of the system suitability test solution, for verification of the specificity of the detection method, the resolution between the main peak of the diiodide and each analyte is not less than 1.5.

[0062] In a preferred embodiment, for the verification of the linear range of the detection method, the diiodide standard has a linear correlation coefficient R ≥ 99.9% within the working concentration range of 50% to 150%, preferably, the linear correlation coefficient R = 1.

[0063] In a preferred embodiment, for the verification of the precision of the detection method in the system adaptability test solution, the relative standard deviation of the diiodide content in 6 solutions is ≤2%, preferably ≤1.31%.

[0064] In a preferred embodiment, for the verification of the precision of the detection method in the system adaptability test solution, the relative standard deviation of the diiodide content in 12 solutions is ≤3%, preferably ≤2.14%.

[0065] In a preferred embodiment, for verification of the accuracy of the detection method, the diiodide has a recovery rate of 98% to 101% within the working concentration range of 50% to 150%, preferably 99.98% to 100.31%.

[0066] Unless otherwise specified, all chemical substances used in the embodiments of this invention are of conventional reagent chemical purity level.

[0067] The technical solution of the present invention will be further described below with reference to specific embodiments:

[0068] Example 1: Preparation of compounds with formula (II)

[0069] The synthetic route for compound (II) is shown below:

[0070]

[0071] (1) Preparation of compounds with structure (I) (CN102093226B Example 1)

[0072] The structural formula of equation (I) is shown below:

[0073]

[0074] Take 1000 mL of acrylonitrile and add 0.25 g of FeCl3 and 0.22 g of p-methoxyphenol at 215 °C and nitrogen pressure of 17 bar. Mix and react for 8 h. After the reaction is complete, 100 mL of dark red liquid is obtained. After distillation at 175 °C, 62 g of crude diacrylcyclobutane is obtained with a cis:trans ratio of approximately 4:6. Then, the crude product is further distilled at 150 °C to obtain 37 g of trans-diacrylcyclobutane with a melting point of 37.5 °C.

[0075] After ammonia gas was introduced into trans-diacyanocyclobutane, the temperature was raised to 80°C, hydrogen gas was pressurized to 20 bar, active nickel was used as catalyst, and the reaction time was 20 h, resulting in 79 mL of light yellow liquid, namely trans-diaaminomethylcyclobutane.

[0076] 79 mL of trans-diaminomethylcyclobutane and 17.5 g of anhydrous oxalic acid were stirred at 70 °C and atmospheric pressure for 3.5 h to form 16.1 g of crude diaminomethylcyclobutane oxalate. The crude product was then dissolved in methanol and purified water at a volume ratio of 5:2 at 70 °C. After complete dissolution, the mixture was cooled and crystallized at 4–6 °C for at least 6 h. The crystals were then filtered and dried to obtain 15.1 g of purified oxalate. This process was repeated twice.

[0077] (2) Preparation of compounds with structure II (refer to page 6,

[0046] -

[0057] of the specification in CN111721843A)

[0078] The compound of formula I (30.0 g, 101.9 mmol), potassium chloroplatinate (36.0 g, 86.7 mmol), potassium iodide (86.0 g, 518.1 mmol), and potassium hydroxide (24.0 g, 427.7 mmol) were dissolved in 170 mL, 180 mL, 87 mL, and 120 mL of purified water, respectively, to obtain solutions A, B, C, and D.

[0079] Heat liquid B to 30°C; stir and break up material A.

[0080] Add solution C to solution B and stir for 0.5 hours to obtain solution E.

[0081] Add solution D to solution A, stir, and the system becomes clear. Filter using a 0.45-micron filter membrane to obtain solution F.

[0082] Add liquid F to liquid E, and a yellow solid precipitates out. Continue stirring at 30°C for 2 hours.

[0083] The filter cake was filtered and washed with purified water (100 mL * 6) until no halogen ions remained. The filter cake was then dried using a rotary evaporator to obtain a crude product (34.0 g) of the compound with structure (II) as a yellow powder.

[0084] Example 2-1: Method for detecting the content of compounds with formula (II)

[0085] Chromatographic conditions and system suitability test

[0086] Blank solvent: N,N-dimethylformamide (DMF).

[0087] Preparation of the test solution: Accurately measure 0.01043 g of trans-1,2-diaminocyclobutane diiodoplatinum(II) test sample (prepared in Example 1) into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take 5 mL of the above solution into a 10 mL volumetric flask and dilute to the mark with DMF; this is denoted as SPL-1.

[0088] Preparation of System Adaptability Solution: Accurately measure 0.01250 g of trans-1,2-diaminocyclobutane diiodoplatinum(II) reference standard (manufacturer: Tianjin WuXi AppTec New Drug Development Co., Ltd.) into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution and add it to a 25 mL volumetric flask, then dilute to the mark with DMF. This solution is designated as STD-1.

[0089] Accurately measure 0.01242 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution into a 25 mL volumetric flask and dilute to the mark with DMF. This solution is designated as STD-2.

[0090] The table below shows the HPLC chromatographic conditions for the determination of the lobaplatin intermediate trans-1,2-diaminocyclobutane diiodoplatin(II).

[0091] Table 1 Chromatographic conditions for liquid chromatography

[0092]

[0093] Assay: Inject 10 μL each of the system suitability test solution and the test solution into the liquid chromatograph and record the chromatograms. The liquid chromatogram of the lobaplatin intermediate is shown below. Figure 12 As shown in a, from Figure 12 As can be seen from a, the retention time of diiodide is 16.865 min, the peak area is 1876.68, and the content of diiodide in the test sample is 38.53%.

[0094] The content of diiodide is calculated according to the following formula:

[0095]

[0096] Among them, V SPL The dilution volume (mL) of the test solution; A SPL The peak area of ​​the test solution; W SPL The sample weight (g) refers to the weight of the test sample; W s The sample weight of the reference standard (average sample weight of STD-1 and STD-2, 0.01246 g); P s The content of the reference standard (identified by quantitative nuclear magnetic resonance (QNMR), the content of diiodide in the reference standard is 99.26%); A s The peak area refers to the average peak area of ​​the reference solution (STD-1 and STD-2, 2310.74); V s This refers to the dilution volume (mL) of the reference solution.

[0097] Example 2-2: Method for detecting the content of compounds with Formula II

[0098] The preparation methods for the blank solvent and the reference solution are exactly the same as those in Example 2-1, wherein the content of diiodide in the reference is 100%; the test sample is prepared by the preparation method of step (2) in Example 1 using diaminomethylcyclobutane oxalate as raw material (manufacturer / seller: Wuhan WuXi AppTec New Drug Development Co., Ltd.), and the remaining preparation steps are exactly the same as those in Example 2-1.

[0099] The table below shows the HPLC chromatographic conditions for the determination of the lobaplatin intermediate trans-1,2-diaminocyclobutane diiodoplatin(II).

[0100] Table 2 Chromatographic conditions for liquid chromatography

[0101]

[0102] Assay: Inject 10 μL each of the system suitability test solution and the test solution into the liquid chromatograph, record the chromatograms, and perform six parallel determinations of the reference solution (equivalent to the system suitability solution). The retention time should be around 15.15 min, and the average peak area of ​​the diiodide peak should be 27.6003 (average peak area of ​​STD-1 and STD-2). The average sample weight of STD-1 and STD-2 is 0.012595 g. The liquid chromatogram of the test sample is shown below. Figure 1 As shown, the retention time of the lobaplatin intermediate was 15.155 min, and the peak area was 37.9402. The content of trans-1,2-diaminocyclobutane diiodoplatin(II) in the test sample was 69.11%.

[0103] Examples 2-3: Methods for detecting the content of compounds with Formula II

[0104] The preparation methods for blank solvent, test solution, and reference solution are exactly the same as in Examples 2-2.

[0105] The table below shows the HPLC chromatographic conditions for the determination of the lobaplatin intermediate trans-1,2-diaminocyclobutane diiodoplatin(II).

[0106] Table 3 Chromatographic conditions for liquid chromatography

[0107]

[0108] Assay: Inject 10 μL each of the system suitability test solution and the test solution into the liquid chromatograph, record the chromatograms, and perform the assay six times in parallel. The retention time should be around 17.37 min. The average peak area of ​​the diiodide peak is 46.46705 (average peak area of ​​STD-1 and STD-2), and the average sample weight of STD-1 and STD-2 is 0.012595 g. The liquid chromatogram of the test sample is shown below. Figure 2 As shown in the figure, the retention time of the lobaplatin intermediate was 17.368 min and the peak area was 65.2959. The content of trans-1,2-diaminocyclobutane diiodoplatin(II) in the test sample was 68.34%.

[0109] Examples 2-4: Methods for detecting the content of compounds with Formula II

[0110] The preparation of blank solvent, test solution and reference solution is exactly the same as in Example 2-2.

[0111] The table below shows the HPLC chromatographic conditions for the determination of the lobaplatin intermediate trans-1,2-diaminocyclobutane diiodoplatin(II).

[0112] Table 4 Chromatographic conditions for liquid chromatography

[0113]

[0114]

[0115] Assay: Inject 10 μL each of the system suitability test solution and the test solution into the liquid chromatograph, record the chromatograms, and perform the assay six times in parallel. The retention time should be around 15.915 min. The average peak area of ​​the diiodide peak is 33.6709 g (average peak area of ​​STD-1 and STD-2). The average sample weight of STD-1 and STD-2 is 0.012595 g. The liquid chromatogram of the test sample is shown below. Figure 3 As shown in the figure, the retention time of the lobaplatin intermediate was 15.910 min, and the peak area was 49.3010. The content of trans-1,2-diaminocyclobutane diiodoplatin(II) in the test sample was 69.01%.

[0116] Example 3: Methodological Validation of the Detection Method

[0117] To verify the practicality and accuracy of this detection method, the following sections describe its specificity, linear range, precision (repeatability, intermediate precision), accuracy, solution stability, and robustness testing:

[0118] 1. Exclusivity

[0119] Blank solvent: N,N-dimethylformamide (DMF).

[0120] Preparation of the test solution: Accurately measure 0.01055 g of trans-1,2-diaminocyclobutane diiodoplatinum(II) test sample into a 25 mL volumetric flask, add approximately 20 mL of DMF to dissolve it, and then dilute to the mark with DMF and mix well. Take 5 mL of the above solution into a 10 mL volumetric flask and dilute to the mark with DMF, denoted as SPL-1.

[0121] Preparation of system adaptability solution: Accurately measure 0.01250 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask, add approximately 20 mL of DMF to dissolve it, and then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution into a 25 mL volumetric flask and dilute to the mark with DMF; this is denoted as STD-1.

[0122] Accurately measure 0.01242 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution into a 25 mL volumetric flask and dilute to the mark with DMF. This solution is designated as STD-2.

[0123] Accurately measure 0.01244 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution and add it to a 25 mL volumetric flask, dilute to the mark with DMF, and label this as STD-3. The resulting STD-3 (Bracket) reference solution is labeled as STD-3 (bracket).

[0124] The STD-1 reference solution was tested six times, with sample names STD-11, STD-13, STD-14, STD-15, and STD-16, and the corresponding liquid chromatograms were as follows: Figure 4 a, Figure 4 b、 Figure 4 c. Figure 4 d、 Figure 4 e and Figure 4 f; The STD-2 reference solution was tested once, and the corresponding liquid chromatograms were as follows: Figure 5 The STD-3 (bracket) reference solution was tested once, and the corresponding liquid chromatograms were as follows: Figure 6 The test results for system adaptability and specificity are shown in the table below:

[0125] Table 5 Test results of system adaptability

[0126]

[0127] The calculation results are shown below:

[0128] ①The average area of ​​the main peaks in the first six STD-1 needles (peaks with retention times around 16.8) is 2309.29;

[0129] ②Relative standard deviation (RSD) of the main peak area of ​​the first six STD-1 injections: 0.15%;

[0130] ③ The recovery rate of the STD-2 reference solution relative to the mean of STD-1 was 100.75%;

[0131] ④ The recovery rate of the STD-3 reference solution relative to the mean of STD-1 was 97.97%.

[0132] The formula for calculating the recovery rate of the reference standard is shown below:

[0133]

[0134] Among them, C s For system suitability, the STD-1 concentration of the solution; A s The average peak area of ​​the first six injections of the system suitability solution STD-1; C x A represents the concentration of the reference standard to be tested. x This represents the peak area of ​​the reference standard to be tested.

[0135] Table 6. Test results of specificity

[0136]

[0137] Note: Blank sample refers to DMF solution, STD-1 refers to the above-mentioned STD-1 reference solution, and SPL refers to SPL-1 test solution.

[0138] In summary, under the above chromatographic conditions, the blank baseline in the obtained chromatogram is clean and stable, with no interference at the main peak position. The resolution between the main peak and each analyte in the solution is not less than 1.5, and the specificity of the method for determining diiodide content meets the requirements.

[0139] 2. Solution stability

[0140] Solution preparation:

[0141] Stability test solution for the test sample: The system suitability solution SPL-1 was used as the stability test solution for the test sample, and data were collected at different time points in this sequence to examine the stability of the test sample solution at 0h, 4h, 8.5h and 14h.

[0142] Stability testing of the reference solution: System suitability solution STD-1 was used as the reference solution for stability testing. Data was collected at different time points in this sequence to examine the stability of the reference solution at 0h, 3h, and 4.5h. This solution was then used as the bracket solution in the next sequence.

[0143] Calculate the diiodide peak area T in the chromatogram for each time interval. x The percentage S of the diiodide peak area T0 in the first injection chromatogram is as follows:

[0144]

[0145] The verification results are as follows:

[0146] Table 7 Stability data of the test solution

[0147] Injection interval (h) Attached Figure Description Main peak area T S 0h Figure 8 a 1944.21 / 4h Figure 8 b 1945.28 100.06％ 8.5h Figure 8 c 1926.23 99.08％ 14h Figure 8 d 2111.99 108.63％

[0148] The results showed that after the diiodide test solution was placed at room temperature for 8.5 h, the percentage S of the diiodide content in the chromatogram of each time interval compared with the diiodide content in the first injection chromatogram was between 98% and 102%, indicating that the diiodide test solution was stable within 8.5 h.

[0149] Table 8 Stability data of reference solution

[0150] Injection interval (h) Attached Figure Main peak area T S 0h Figure 9 a 2306.89 / 3h Figure 9 b 2306.34 99.98％ 4.5h Figure 9 c 2235.87 96.92％

[0151] The results showed that after the diiodide reference solution was placed at room temperature for 3 hours, the percentage S of the diiodide content in the chromatogram of each time interval compared with the diiodide content in the first injection chromatogram was between 98% and 102%, indicating that the diiodide test solution was stable within 3 hours.

[0152] 3. Linearity and Range

[0153] Prepare a set of linear solutions with nominal concentrations of 50%, 75%, 100%, 125%, and 150% (0.1 mg / mL), each containing approximately 0.05 mg / mL, 0.075 mg / mL, 0.1 mg / mL, 0.125 mg / mL, and 0.15 mg / mL of trans-1,2-diaminomethylcyclobutane diiodide platinum(II) reference standard, respectively.

[0154] Preparation of solution: Accurately weigh 0.01265 g of trans-1,2-diaminomethylcyclobutane diiodide platinum(II) reference standard, transfer it to a 50 mL volumetric flask, add about 20 mL of DMF to dissolve it, dilute to the mark with DMF, shake well, and label it STOCK-1 (reference standard concentration is 0.253 mg / mL).

[0155] L-150: Accurately transfer 6 mL of STOCK-1 solution into a 10 mL volumetric flask, dilute to the mark with DMF, and shake well.

[0156] L-125: Accurately transfer 5 mL of STOCK-1 solution into a 10 mL volumetric flask, dilute to the mark with DMF, and shake well.

[0157] L-100: Accurately transfer 4 mL of STOCK-1 solution into a 10 mL volumetric flask, dilute to the mark with DMF, and shake well.

[0158] L-75: Accurately transfer 3 mL of STOCK-1 solution into a 10 mL volumetric flask, dilute to the mark with DMF, and shake well.

[0159] L-50: Accurately transfer 2 mL of STOCK-1 solution into a 10 mL volumetric flask, dilute to the mark with DMF, and shake well.

[0160] One injection was given for each concentration level of the linear solution.

[0161] Within the concentration range of 50% to 150%, a graph was plotted with compound concentration on the x-axis and peak area response on the y-axis to obtain the linear equation and correlation coefficient R for the diiodine compound, as shown below. Figure 10 As shown in the table below:

[0162] Table 9 Linear Data

[0163]

[0164] Note: The nominal concentration response is the ratio of the peak area (L-100) at 100% limit concentration to its concentration.

[0165] In summary, diiodide showed good linearity in the range of 0.0506 mg / mL to 0.1518 mg / mL, with a linear correlation coefficient of 1.

[0166] 4. Accuracy

[0167] The five reference solutions L-50, L-75, L-100, L-125, and L-150 from the linearity and range validation test were used as the test reference solutions. The recovery rate was calculated using the mean of the six STD-1 injections. The formula for calculating the recovery rate is shown below:

[0168]

[0169] The formula for calculating concentration is as follows:

[0170]

[0171] Where: ms refers to the sample weight of the system adaptability control solution STD-1, in mg; AL-i refers to the peak area at each linear concentration; AS refers to the mean of the six peak areas of the system adaptability STD-1; VS refers to the dilution volume of the system adaptability control solution STD-1.

[0172] The formula for calculating the concentration is as follows:

[0173]

[0174] The verification results are shown in Table 10 below:

[0175] Table 10 Accuracy

[0176]

[0177] In summary, the recovery rate of the diiodide test solution was 99.98%-100.31% within the working concentration range of 50%-150%, indicating that the method for determining diiodide content has good accuracy.

[0178] 5. Precision

[0179] (1) Preparation of repeatable solutions (Experimenter A):

[0180] Prepare a nominal concentration (0.2 mg / mL) test solution: Accurately weigh six portions of trans-1,2-diaminomethylcyclobutane diiodide platinum(II) test sample, namely 0.01043 g, 0.01041 g, 0.01030 g, 0.01025 g, 0.01017 g, and 0.01040 g. Place each portion into six 25 mL volumetric flasks, add approximately 20 mL of DMF to dissolve, and then dilute to the mark with DMF and mix well. Take 5 mL of each of the above solutions into six 10 mL volumetric flasks and dilute to the mark with DMF. Label as SPL-1-A to SPL-6-A.

[0181] Preparation of system adaptability solution: Accurately measure 0.01250 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask, add approximately 20 mL of DMF to dissolve it, and then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution into a 25 mL volumetric flask and dilute to the mark with DMF; this is denoted as STD-1.

[0182] Accurately measure 0.01242 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution into a 25 mL volumetric flask and dilute to the mark with DMF. This solution is designated as STD-2.

[0183] Accurately measure 0.01244 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution and add it to a 25 mL volumetric flask, dilute to the mark with DMF, and label this as STD-3. This yields the STD-3 (Bracket) reference solution, labeled as STD-3 (bracket). This serves as the last Bracket solution in this sequence.

[0184] Testing instrument: Agilent-1260HPLC-026; Column: Waters-XSelect-HSS T3 (150*4.6mm3.5μm) Column-133.

[0185] (2) Preparation of intermediate precision solution (Experimenter B):

[0186] Six test solutions and system suitability solutions were prepared by different technicians at different times.

[0187] Prepare a nominal concentration (0.2 mg / mL) test solution: Accurately weigh six portions of trans-1,2-diaminomethylcyclobutane diiodide platinum(II) test sample, namely 0.01047 g, 0.01036 g, 0.01012 g, 0.01032 g, 0.01052 g, and 0.01020 g. Place each portion into six 25 mL volumetric flasks, add approximately 20 mL of DMF to dissolve, and then dilute to the mark with DMF. Mix well. Take 5 mL of each solution and place it into six 10 mL volumetric flasks, and dilute to the mark with DMF. Label as SPL-1-B to SPL-6-B.

[0188] Preparation of system adaptability solution: Accurately measure 0.01224 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask, add approximately 20 mL of DMF to dissolve it, and then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution into a 25 mL volumetric flask and dilute to the mark with DMF; this is denoted as STD-1.

[0189] Accurately measure 0.01231 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution into a 25 mL volumetric flask and dilute to the mark with DMF. This solution is labeled STD-2.

[0190] Accurately measure 0.01230 g of trans-1,2-diaminocyclobutane diiodoplatin(II) reference standard into a 25 mL volumetric flask. Add approximately 20 mL of DMF to dissolve it, then dilute to the mark with DMF and mix well. Take another 5 mL of the above solution and add it to a 25 mL volumetric flask, dilute to the mark with DMF, and label this as STD-3. This yields the STD-3 (Bracket) reference solution, labeled as STD-3 (bracket). This serves as the last Bracket solution in this sequence.

[0191] Testing instruments: Shimadzu LC-20AD SHIMADZU HPLC-029; Column: Waters-XSelect-HSS T3 (150*4.6mm 3.5μm) Column-95.

[0192] The repeatability results and intermediate precision results are shown in Tables 11 and 12, respectively.

[0193] Table 11 Repeatability Test Results

[0194]

[0195] Table 12 Test Results of Intermediate Precision

[0196]

[0197]

[0198] In summary, the RSD of the content of six diiodide solutions was 1.31%, and the RSD of the diiodide content was 0.68% after changing the tester. The RSD of the content of 12 diiodide test solutions was 2.14%, indicating that the precision of diiodide is good under this method.

[0199] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting the content of a lobaplatin intermediate, characterized in that, The lobortanium intermediate is trans-1, 2-diaminocyclobutane diiodoplatinum (II), and its structural formula is as follows: ； The detection method is an HPLC method, and the detection conditions of the HPLC method are as follows: octadecylsilane bonded silica gel is used as a filler, an ammonium acetate solution is used as a mobile phase A, and a mixed solution of the ammonium acetate solution and acetonitrile is used as a mobile phase B, the volume ratio of the ammonium acetate solution to acetonitrile in the mobile phase B is 1: (5-19), and gradient elution is performed; the gradient elution mode is as follows: 0-25 minutes: the mobile phase A is reduced from 100% to 10% by volume, and the mobile phase B is increased from 0% to 90% by volume; The preparation method of the lobortanium intermediate comprises the following steps: (1) Preparation of a compound with a structure of formula (I): The structural formula of formula (I) is as follows: Take 1000 mL of acrylonitrile, add 0.25 g of FeC13 and 0.22 g of p-methoxyphenol under the conditions of a temperature of 215 DEG C and nitrogen pressurization to 17 bar, mix and react for 8 h, after the reaction is completed, 100 mL of a deep red liquid is obtained, after distillation at 175 DEG C, 62 g of a dicyanocyclobutane crude product is obtained, the cis-trans ratio of which is about 4:6, then the crude product is subjected to rectification at 150 DEG C to obtain 37 g of trans-dicyanocyclobutane, and the melting point is 37.5 DEG C; After ammonia gas is introduced into the trans-dicyanocyclobutane, the temperature is increased to 80 DEG C, hydrogen is pressurized to 20 bar, active nickel is used for catalysis, and the reaction time is 20 h, so that 79 mL of a light yellow liquid, i.e., trans-diaminomethylcyclobutane, is obtained; Take 79 mL of the trans-diaminomethylcyclobutane and 17.5 g of anhydrous oxalic acid, stir at 70 DEG C under normal pressure for 3.5 h, combine to form 16.1 g of a diamino-methylcyclobutane oxalate crude product, then dissolve the prepared crude product with methanol and purified water in a volume ratio of 5:2 at 70 DEG C, after complete dissolution, cool and crystallize at 4-6 DEG C for more than 6 h, filter and dry to obtain a diamino-methylcyclobutane oxalate fine product; (2) Preparation of trans-1, 2-diaminocyclobutane diiodoplatinum (II): Dissolve 101.9 mmol of the compound with the structure of formula (I), 86.7 mmol of potassium chloroplatinite, 518.1 mmol of potassium iodide and 427.7 mmol of potassium hydroxide in 170 mL, 180 mL, 87 mL and 120 mL of purified water respectively to obtain A, B, C and D liquids; Warm the B liquid to 30 DEG C; stir and disperse the A material; Add the C liquid to the B liquid, stir for 0.5 h to obtain an E liquid; Add the D liquid to the A liquid, stir, the system becomes clear, filter with a 0.45-micron filter membrane to obtain an F liquid; Add the F liquid to the E liquid, yellow solid is precipitated, continue to stir at 30 DEG C for 2 h; Filter, wash the filter cake with purified water until no halogen ions are left, dry the filter cake with a rotary evaporator to obtain a yellow compound crude product.

2. The method of claim 1, wherein the intermediate content of lobaplatin is detected by the method comprising: The concentration of the ammonium acetate solution in the mobile phase A is 8-12 mmol / L.

3. The method of claim 1, wherein the intermediate content of lobaplatin is detected by the method comprising: The concentration of the ammonium acetate solution in the mobile phase A is 9-11 mmol / L.

4. The method of claim 1, wherein the intermediate is a compound of formula (II): ###0002### (II) and the method comprises detecting the presence of the compound of formula (II) in the sample. The volume ratio of the ammonium acetate solution to acetonitrile is 1: (5-15).

5. The method of claim 1, wherein the intermediate content of lobaplatin is detected by HPLC. The concentration of the ammonium acetate solution in the mobile phase B is 8-12 mmol / L.

6. The method of claim 2, wherein the intermediate is a compound of formula (II): ###0002### (II) and the method comprises detecting the presence of the compound of formula (II) in the sample. The concentration of the ammonium acetate solution in the mobile phase B is 8-12 mmol / L.

7. The method of claim 1, wherein the intermediate content of lobaplatin is detected by HPLC. The concentration of the ammonium acetate solution in the mobile phase B is 9-11 mmol / L.

8. The method of claim 1-7, wherein, The method comprises the following steps: (1) Preparation of the control solution The trans-1, 2-diaminocyclobutane platinum diiodide control sample is weighed and dispersed in N, N-dimethylformamide; (2) Preparation of the test solution The trans-1, 2-diaminocyclobutane platinum diiodide test sample is weighed and dispersed in N, N-dimethylformamide; (3) High performance liquid chromatography analysis The gradient elution mode of the high performance liquid chromatography analysis is as follows: 0-25 minutes: the mobile phase A is reduced from 100% to 10%, and the mobile phase B is increased from 0% to 90%.

9. The method of claim 8, wherein the intermediate content of lobaplatin is detected by HPLC. The gradient elution time in the gradient elution mode is increased by 2-3 minutes.

10. The method of claim 8, wherein the intermediate content of lobaplatin is detected by HPLC. The gradient elution time in the gradient elution mode is reduced by 2-3 minutes.

11. The method of claim 1-7, wherein the detection of the intermediate content of lobaplatin is performed by HPLC. The flow rate in the detection condition of the HPLC method is 0.8-1.2 mL / min; and / or, the column temperature is 30-45℃; and / or, the detection wavelength is 219-221 nm.

12. The method of claim 8, wherein the intermediate content of lobaplatin is detected by HPLC. The flow rate in the detection condition of the HPLC method is 0.8-1.2 mL / min; and / or, the column temperature is 30-45℃; and / or, the detection wavelength is 219-221 nm.

13. The method of claim 9, wherein the intermediate content of lobaplatin is detected by HPLC. The flow rate in the detection condition of the HPLC method is 0.8-1.2 mL / min; and / or, the column temperature is 30-45℃; and / or, the detection wavelength is 219-221 nm.

14. The method of claim 10, wherein the intermediate content of lobaplatin is detected by HPLC. The flow rate in the detection condition of the HPLC method is 0.8-1.2 mL / min; and / or, the column temperature is 30-45℃; and / or, the detection wavelength is 219-221 nm.

15. The method of claim 1-7, wherein the detection of the intermediate content of lobaplatin is performed by HPLC. The separation degree of the main peak of the compound (II) from each to-be-detected impurity in the chromatogram of the system suitability test solution is not less than 1.

5.

16. The method of claim 8, wherein the intermediate content of lobaplatin is detected by HPLC. The separation degree of the main peak of the compound (II) from each to-be-detected impurity in the chromatogram of the system suitability test solution is not less than 1.

5.

17. The method of claim 9, wherein the intermediate content of lobaplatin is detected by HPLC. The separation degree of the main peak of the compound (II) from each to-be-detected impurity in the chromatogram of the system suitability test solution is not less than 1.

5.

18. The method of claim 10, wherein, The separation degree of the main peak of the compound (II) from each to-be-detected impurity in the chromatogram of the system suitability test solution is not less than 1.

5.

19. The method of claim 11, wherein the intermediate content of lobaplatin is detected by HPLC. The separation degree of the main peak of the compound (II) from each to-be-detected impurity in the chromatogram of the system suitability test solution is not less than 1.

5.

20. The method for detecting the content of a lobaplatin intermediate according to any one of claims 1-19 is used for the quality control of a lobaplatin intermediate bulk drug or preparation.

Images