A method for determining the content of non-deuterated TSL-0202 in TSL-0202

Abstract

The invention relates to a method for determining the content of non-deuterated TSL-0202 in TSL-0202. The method comprises the following steps: step 1, preparing a test solution: firstly preparing an internal standard solution containing a deuterium compound, dissolving a test sample TSL-0202 with the internal standard solution, and preparing a test solution with a determined concentration; step 2, loading the test solution into a nuclear magnetic resonance instrument for determining the type and number of hydrogen atoms in a molecule for detection, and obtaining a nuclear magnetic resonance spectrum of the test sample; and step 3, calculating the content of non-deuterated TSL-0202 in the test sample TSL-0202 according to the nuclear magnetic resonance spectrum.

Description

Technical Field

[0001] The present invention relates to a method for determining the content of a drug compound, in particular to a method for determining the content of non-deuterated TSL-0202 in TSL-0202. Background Art

[0002] Thrombosis refers to the formation of clots in the blood flowing in the blood vessel cavity or heart, thereby blocking the blood vessel cavity, causing ischemia, hypoxia and necrosis of myocardial, brain and other tissues. Thrombosis can occur in blood vessels in any part of the human body, causing myocardial infarction, stroke, cerebral infarction, pulmonary embolism, and is also a common complication in surgical operations and the cause of reocclusion after interventional angioplasty. It has a high disability and mortality rate. Thrombosis can occur at any age and at any time, especially venous thrombosis, which is the root cause of the top three fatal vascular diseases in the world (heart disease, stroke, venous thromboembolism). One to three people in every 1,000 people in the general population suffer from the disease. There are nearly 10 million cases of thrombotic diseases worldwide each year, and one in every four deaths is related to thrombosis. Thrombotic diseases affect a wide range of people, and the drug market is growing rapidly. Among them, antiplatelet drugs are the best performing antithrombotic drugs in the global market.

[0003] P2Y12 inhibitors are a class of antiplatelet drugs that act on platelet P2Y12 receptors and can inhibit platelet aggregation caused by adenosine diphosphate (ADP). They are mainly used clinically to prevent and treat thrombotic events in cardiovascular diseases. The dual antiplatelet therapy (DAPT) regimen of P2Y12 inhibitors combined with aspirin is a commonly used antithrombotic treatment regimen for cardiovascular diseases recommended by various guidelines. Currently, the P2Y12 inhibitors available for clinical use include clopidogrel, prasugrel, and ticagrelor.

[0004] Among them, clopidogrel is the P2Y12 inhibitor with the widest clinical application, the longest time and the most research evidence, which is mainly due to its higher drug safety and lower bleeding risk. Since its launch in 1997, clopidogrel has accumulated sufficient evidence-based medicine evidence in antiplatelet strategies and has always been a Class I recommended drug in various guidelines. Despite the emergence of new antiplatelet drugs, clopidogrel remains the cornerstone drug for antiplatelet therapy with its comprehensive, strong evidence-based medicine evidence and rich clinical application experience.

[0005]

[0006] However, clopidogrel is a prodrug that needs to be metabolized by liver CYP450 enzymes (CYP2C19, CYP3A4, etc.) to form active products. However, due to CYP2C19 enzyme gene polymorphism, some patients (>30%) cannot effectively metabolize clopidogrel into active ingredients, resulting in the phenomenon of "clopidogrel resistance" (Circulation. 2004; 109: 166-171). In order to avoid "clopidogrel resistance", a variety of P2Y12 inhibitors with different skeletons are currently on the market or under development, but they all have bleeding and carcinogenic risks, or side effects such as drug off-target, which are greatly limited in clinical application.

[0007] Chinese patent CN201310428052.4 discloses a series of clopidogrel metabolites, 2-oxyclopidogrel prodrug thieno-piperidine derivatives, which are used to improve "clopidogrel resistance". However, this series of compounds still has defects such as low platelet aggregation inhibition rate and fast hydrolysis rate. In order to solve these defects, develop new antiplatelet aggregation drugs with rapid clinical onset, high efficacy, improved bioavailability, reduced side effects, and easy dissolution, absorption, and administration, Chinese patent CN201680036795.9 discloses a series of novel deuterated thieno-piperidine derivatives based on CN201310428052.4. Among them, a deuterated compound TSL-0202 is disclosed, with the chemical name: (S)-2-(2-chlorophenyl)-2-(2-((dimethoxyphosphoryl)oxy)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-yl)acetic acid trideuterium methyl ester. The structural formula is as follows:

[0008]

[0009] This compound can be directly hydrolyzed into the precursor of the active drug of clopidogrel without relying on the activity of CYP2C19 esterase. At the same time, the final active metabolite is consistent with clopidogrel, so it can retain the good safety characteristics of clopidogrel while avoiding the problem of "clopidogrel resistance" caused by the weak metabolism of CYP2C19 enzyme.

[0010] However, in the process of synthesizing TSL-0202, the deuterated reagent used is deuterated methanol, which contains methanol. Therefore, the by-product of TSL-0202 is a non-deuterated product. It is necessary to control the content of the non-deuterated product to ensure the yield of TSL-0202. There is currently no literature report on the determination of non-deuterated TSL-0202 content in TSL-0202. Summary of the invention

[0011] The present invention adopts quantitative nuclear magnetic resonance technology (Quantitative Nuclear Magnetic Resonance, referred to as QNMR) to measure the content of non-deuterated TSL-0202 in TSL-0202, and the nuclear magnetic resonance instrument used is used to measure the type and number of hydrogen atoms in the molecule.

[0012] The present invention provides a method for determining the content of non-deuterated TSL-0202 in TSL-0202, wherein the non-deuterated TSL-0202 is a substance in which D is H in the structure of TSL-0202.

[0013] The method of the present invention comprises the following steps:

[0014] Step 1, preparation of the test solution: first prepare an internal standard solution containing a deuterium compound, dissolve the test product TSL-0202 with the internal standard solution, and prepare a test solution of a determined concentration;

[0015] Step 2, loading the sample solution into a nuclear magnetic resonance instrument for determining the type and number of hydrogen atoms in a molecule to obtain a nuclear magnetic resonance spectrum of the sample;

[0016] Step 3, according to the nuclear magnetic resonance spectrum, calculate the content of non-deuterated TSL-0202 in the test product TSL-0202.

[0017] The internal standard solution is prepared as follows: weigh an appropriate amount of succinic anhydride, dissolve it in deuterated chloroform, and prepare an internal standard solution; in the internal standard solution, the concentration of the deuterium compound is 0.01 mg / mL-10 mg / mL.

[0018] The test solution is prepared as follows: take an appropriate amount of TSL-0202 raw material, place it in a volumetric bottle, dissolve it with the internal standard solution and dilute it to the scale as the test solution; in the test solution, the concentration of TSL-0202 is 0.01 mg / mL-10 mg / mL.

[0019] The nuclear magnetic resonance instrument can be any type of instrument for measuring the type and number of hydrogen atoms in a molecule, and the magnetic field frequency range is 300MHz-1.2GHz.

[0020] The NMR conditions are: deuterated chloroform as solvent, succinic anhydride as internal standard, and the test conditions are as follows: relaxation time d1 ≥ 10s; scanning number ns ≥ 32 times; empty scan DS = 4 times; spectrum width SW = 18-22PM; sampling time AQ = 3.5-5.5s; center frequency O1 = 2500-2700HZ; measured at room temperature 1 H NMR spectroscopy.

[0021] The determination is to transfer the sample solution into a nuclear magnetic resonance tube, set the nuclear magnetic resonance condition parameters, adjust the speed of the nuclear magnetic resonance tube so that the rotating edge does not interfere with the measured signal, turn on the machine and record the spectrum, preferably measuring 5 times.

[0022] The calculation adopts the integration method to calculate the peak area of ​​each characteristic peak and the peak area of ​​the internal standard peak respectively, and preferably calculates the average value of 5 measurements.

[0023] The content of non-deuterium TSL-0202 in TSL-0202 is further calculated according to the following formula:

[0024]

[0025] Where W r is the weight of the internal standard;

[0026] A s and A r are the average peak areas of the characteristic peaks of the test sample and the internal standard peak, respectively;

[0027] E s and E r are the proton equivalent weight (mass) of the test sample and the internal standard, respectively (calculated by dividing the molecular weight by the number of protons in the characteristic peak)

[0028] Among them, TSL-0202 is self-made.

[0029] Deuterated chloroform is commercially available.

[0030] The specifications of NMR tubes are: outer diameter 4mm-11mm, length 14cm-21cm.

[0031] Preferably, the method of the present invention comprises the following steps:

[0032] (1) Preparation of test solution: Accurately weigh an appropriate amount of succinic anhydride and dissolve it in deuterated chloroform to prepare an internal standard solution with a concentration of about 0.05 mg / ml. Accurately weigh 10 mg of TSL-0202 API and place it in a 1 ml volumetric flask. Dissolve it in the internal standard solution and dilute to the mark as the test solution.

[0033] (2) Determination of non-deuterated TSL-0202 content: Transfer the sample solution into the NMR tube, correctly set the instrument parameters, adjust the NMR tube speed so that the rotating edge does not interfere with the measured signal, and record the spectrum.

[0034] (3) Use the integration method to measure the peak area of ​​the characteristic peak and the peak area of ​​the internal standard peak of each sample, repeat the measurement 5 times, and take the average value. The content of non-deuterated TSL-0202 in TSL-0202 is calculated as follows:

[0035]

[0036] Where W r is the weight of the internal standard;

[0037] A s and A r are the average peak areas of the characteristic peaks of the test sample and the internal standard peak, respectively;

[0038] E s and E r are the proton equivalent weight (mass) of the test sample and the internal standard, respectively (calculated by dividing the molecular weight by the number of protons in the characteristic peak)

[0039] The NMR parameters are as follows: deuterated chloroform as solvent, succinic anhydride as internal standard, and the test conditions are as follows: relaxation time d1 = 10 s; scanning number ns = 32 times; empty scan DS = 4 times; spectrum width SW = 20.0254 PM; sampling time AQ = 4.0894966 s; center frequency O1 = 2600.85 Hz; measured at room temperature 1 H NMR spectroscopy.

[0040] Among them, δ H 3.7427 (3H, S) is the quantitative peak of non-deuterated TSL-0202, δ H 3.0283 (4H, S) is the quantitative peak of succinic anhydride internal standard.

[0041] The experimental conditions of the present invention are obtained by screening:

[0042] 1. Selection of solvent

[0043] In addition to having good solubility for the sample, the residual signal peak of the suitable solvent should not interfere with the signal peak of the analyzed sample and the internal standard. TSL-0202 has good solubility in chloroform, dimethyl sulfoxide and methanol, but the residual proton signal of deuterated methanol and deuterated dimethyl sulfoxide may interfere with the signal peak of the internal standard. Therefore, deuterated chloroform was selected as the solvent.

[0044] 2. Selection of internal standard and quantitative peak

[0045] The internal standard should have accurate content, high purity, stable chemical properties, be easily soluble in the selected solvent, and not react with the sample. The selected internal standard is preferably a sharp single peak containing multiple protons, the quantitative peak of the internal standard does not overlap with the quantitative peak of the sample to be tested, and can be well separated from the sample absorption peak. In this experiment, succinic anhydride is stable in deuterated chloroform, does not react with TSL-0202 and non-deuterated TSL-0202, and has a good separation from the response peak of non-deuterated TSL-0202, so it is selected as the internal standard substance. Therefore, the present invention selects δ H 3.7427 (3H, S) is the quantitative peak of non-deuterated TSL-0202, δH 3.0283 (4H, S) is the quantitative peak of succinic anhydride internal standard.

[0046] 3. Selection of NMR experimental parameters

[0047] 3.1d1 selection

[0048] In the QNMR experiment, d1 (relaxation time) must be set long enough (≥5T1) to allow the nuclei to relax completely, thereby ensuring that the integrated signal intensity is proportional to the number of nuclei. To consider the impact of d1 on the experimental results, for the same sample to be tested, we set d1 to 2s, 5s, 10s, 20s and 30s respectively. The results show that when d1≥10s, the integrated area of ​​the quantitative peak of the sample to be tested and the internal standard no longer changes. In order to simultaneously meet d1≥5T1 and save sampling time, the present invention selects d1 as 10s.

[0049] 3.2ns selection

[0050] The size of ns directly affects the signal-to-noise ratio of the NMR signal: the larger the ns, the higher the signal-to-noise ratio and the more accurate the quantification; conversely, it will affect the accuracy of quantification. For QNMR, S / N≧250 is generally required. However, the larger the ns, the longer the sampling time. We set ns to 2, 4, 8, 16, 32 and 64 respectively. The results show that when ns>16, the signal-to-noise ratio of the sample to be tested and the internal standard NMR quantitative peak has met the quantitative analysis requirements. The present invention selects ns as 32.

[0051] 4. Methodological Validation

[0052] At present, relevant technical specifications for NMR quantitative analysis methods and verification have not been published internationally. Referring to the guidelines in Volume IV of the Chinese Pharmacopoeia and General Chapter 40-761 of the United States Pharmacopoeia, we verified the stability, linear range, limit of quantification, accuracy, precision and durability of this method.

[0053] 4.1 Determination of linear range

[0054] Take 2 mg of succinic anhydride, weigh it accurately, place it in a 2 ml volumetric flask, dissolve it with deuterated chloroform and dilute it to the mark as the internal standard solution stock solution. Accurately pipette 0.5 ml of the internal standard solution stock solution, place it in a 10 ml volumetric flask, dilute it to the mark with deuterated chloroform as the internal standard solution.

[0055] Take 2mg of non-deuterated TSL-0202, weigh accurately, place in a 1ml volumetric bottle, dissolve with internal standard solution and dilute to scale as reference solution stock solution. Accurately pipette 50μl, 65μl, 80μl, 100μl, 120μl of reference solution stock solution into different 1ml volumetric bottles, dilute to scale with internal standard solution as linear relationship solutions 1 to 5.

[0056] Take linear relationship solutions 1-5 and transfer them to different NMR tubes. Measure each concentration once. Use concentration as the abscissa and the ratio of the peak area of ​​the target substance to the peak area of ​​the internal standard substance as the ordinate to establish a linear relationship equation. The results are as follows:

[0057] Table 1: Non-deuterated TSL-0202 linearity results

[0058] Concentration (mg / mL) 0.1094 0.1422 0.1859 0.2187 0.2625 Peak area ratio 0.3251 0.4408 0.5693 0.7196 0.8556

[0059] The results showed that non-deuterated TSL-0202 had a good linear relationship in the concentration range of 0.1094 mg / ml to 0.2625 mg / ml, the linear equation was Y=3.4931X-0.0597, and the correlation coefficient r was 0.9981, which was greater than 0.99, meeting the requirements of the United States Pharmacopoeia 40-761 general chapter.

[0060] 4.2 Accuracy Experiment

[0061] Take 2 mg of succinic anhydride, accurately weigh it, place it in a 2 ml volumetric flask, dissolve it with deuterated chloroform and dilute it to the mark as the internal standard solution stock solution. Accurately pipette 1 ml of the internal standard solution stock solution, place it in a 20 ml volumetric flask, dilute it with deuterated chloroform to the mark as the internal standard solution. Take 4 mg of non-deuterated TSL-0202, accurately weigh it, place it in a 2 ml volumetric flask, dissolve it with the internal standard solution and dilute it to the mark as the reference solution stock solution.

[0062] 50% limit level spike solution: Take about 10 mg of TSL-0202 API, weigh accurately, place in a 1 ml volumetric flask, accurately add 0.05 ml of reference stock solution, dissolve with internal standard solution and dilute to scale to obtain 50% limit level spike solution. Prepare 6 portions in parallel.

[0063] 100% limit level spike solution: Take about 10 mg of TSL-0202 API, weigh accurately, place in a 1 ml volumetric flask, accurately add 0.1 ml of reference stock solution, dissolve with internal standard solution and dilute to scale to obtain 100% limit level spike solution. Prepare 6 portions in parallel.

[0064] 120% limit level spike solution: Take about 10 mg of TSL-0202 API, weigh accurately, place in a 1 ml volumetric flask, accurately add 0.12 ml of reference stock solution, dissolve with internal standard solution and dilute to scale to obtain 120% limit level spike solution. Prepare 3 copies in parallel.

[0065] Test sample (unspecified) solution: Take about 10 mg of TSL-0202 raw material, accurately weigh, place in a 1 ml volumetric flask, dissolve and dilute to the scale with the internal standard solution to obtain the test sample (unspecified) solution.

[0066] Take the test sample (without spike) solution and the above three levels of spiked solutions for injection, calculate the recovery rate, and the results are shown in the following table:

[0067] Table 2: Non-deuterium TSL-0202 recovery test results

[0068]

[0069] The results showed that the average recoveries of the spiked test solution at three concentration levels of 50%, 100%, and 120% were 95.91% (n=6), 100.79% (n=6), and 94.77% (n=3), respectively. The recoveries were all between 80% and 120%, which met the requirements of the United States Pharmacopoeia 40-761.

[0070] 4.3 Limit of quantification

[0071] Refer to the preparation method of "50% limit level solution" under "4.2 Accuracy" and prepare six spiked test samples in parallel. In the six spiked test solutions, the recovery of non-deuterated TSL-0202 is between 70% and 130%, and the RSD is less than 15% (see Table 2), which meets the requirements of the general chapter of USP 40-761. The 50% limit concentration is the limit of quantification (LOQ) concentration, which is equivalent to a sample content of 1.0%.

[0072] Table 3: Limits of Quantitation

[0073]

[0074] Results: The quantitative limit of TSL-0202-ND was 0.1 mg / ml, equivalent to 1.0% of the sample content.

[0075] 4.4 Precision

[0076] 4.4.1 Spiking repeatability test

[0077] According to the preparation method of "100% limit level addition solution" under "4.2 Accuracy", 6 spiked test solutions were prepared in parallel to calculate the recovery rate. The results of the repeatability experiment are shown in Table 3. The RSD value of the recovery rate of non-deuterated TSL-0202 in the 6 spiked repeatable solutions was 1.12%, indicating that the repeatability of the method was good.

[0078] Table 4: Results of the spike repeatability test

[0079]

[0080] 4.4.2 Intermediate precision experiment

[0081] Different personnel prepared 6 spiked samples in parallel according to the preparation method of "100% limit level addition solution" under "4.2 Accuracy". The results are shown in Table 4. The RSD value of the intermediate precision of non-deuterated TSL-0202-recovery rate is 4.50%, which is less than 25%, which meets the requirements of the general chapter of USP 40-761, indicating that the intermediate precision of the method is good.

[0082] Table 5: Intermediate precision test results

[0083]

[0084] 4.5 Stability test

[0085] The spiked test solution was placed at room temperature for 0h, 1h, 2h, 4h, 6h, 8h, 24h, and 48h for NMR experiments. The results showed that the RSD of the peak area ratio of the sample non-deuterated TSL-0202 and succinic anhydride was 0.41% (n=8), indicating that the spiked test solution was stable when placed at room temperature for 48h.

[0086] Table 6: Stability of spiked test solution

[0087]

[0088] 4.6 Durability test

[0089] Durability refers to the degree to which the test results are not affected by slight changes in test conditions. This paper mainly examines the effect of the number of scans on the experimental results. Prepare the 100% limit concentration spiked test sample as described in Section 4.2, and collect the data under the experimental conditions of 16, 32, and 64 scans. 1 HNMR spectrum. The results show that when the sampling times vary from 16 to 64, the number of scans has little effect on the determination results, and the RSD of the non-deuterated TSL-0202 content in the spiked sample is 0.96%, indicating that the method has good durability.

[0090] Table 7: Parameters of NMR acquisition condition changes

[0091]

[0092] Table 8: Durability test results of spiked test solution

[0093] H NMR spectroscopy parameters Non-deuterium TSL-0202 content (%) Scan times 16-1 2.2823 Scan times 16-2 2.2925 Scan times 16-3 2.2772 Scan times 16-4 2.3268 Scan times 16-5 2.2800 Scan times 16-6 2.2638 Scan times 32-1 2.3410 Scan times 32-2 2.3004 Scan times 32-3 2.3271 Scan times 32-4 2.2947 Scan times 32-5 2.3000 Scan times 32-6 2.2621 Scan times 64-1 2.3124 Scan times 64-2 2.2951 Scan times 64-3 2.2861 Scan times 64-4 2.3227 Scan times 64-5 2.2962 Scan times 64-6 2.3129 RSD(%) 0.96

[0094] 4.7 Exclusivity

[0095] Take 2 mg of succinic anhydride, accurately weigh it, place it in a 2 ml volumetric flask, dissolve it with deuterated chloroform and dilute it to the mark as the internal standard solution stock solution. Accurately pipette 1 ml of the internal standard solution stock solution, place it in a 20 ml volumetric flask, dilute it with deuterated chloroform to the mark as the internal standard solution. Take 4 mg of non-deuterated TSL-0202, accurately weigh it, place it in a 2 ml volumetric flask, dissolve it with the internal standard solution and dilute it to the mark as the reference solution stock solution. Accurately pipette 0.1 ml of the reference solution stock solution into a 1 ml volumetric flask, dilute it with the internal standard solution to the mark to obtain the reference solution.

[0096] Take about 10 mg of TSL-0202 raw material, accurately weigh it, put it in a 1 ml volumetric flask, dissolve it with the internal standard solution and dilute it to the scale to obtain the test solution.

[0097] Take about 10 mg of TSL-0202 raw material, accurately weigh it, put it in a 1 ml volumetric flask, accurately add 0.1 ml of reference substance stock solution, dissolve it with internal standard solution and dilute it to the scale to obtain the spiked test solution.

[0098] Transfer the blank solvent, control solution, test solution, and spiked test solution into NMR tubes for determination.

[0099] Table 9: Reference solution

[0100] name Non-deuterated TSL-0202 Succinic anhydride internal standard Displacement δ3.7450(3H,S) δ3.0326(4H,S)

[0101] Results: There was no obvious interference between the position of the analyte and the position of the internal standard substance in the blank solution and the test solution, and the method had good specificity

[0102] 4.8 Instrument Precision

[0103] Take 2 mg of succinic anhydride, accurately weigh it, place it in a 2 ml volumetric flask, dissolve it with deuterated chloroform and dilute it to the scale as the internal standard solution stock solution. Accurately pipette 0.25 ml of the internal standard solution stock solution, place it in a 5 ml volumetric flask, dilute it to the scale with deuterated chloroform as the internal standard solution. Take 2 mg of non-deuterated TSL-0202, accurately weigh it, place it in a 1 ml volumetric flask, dissolve it with the internal standard solution and dilute it to the scale as the reference solution stock solution. Accurately pipette 0.1 ml of the reference solution stock solution into a 1 ml volumetric flask, dilute it to the scale with the internal standard solution to obtain the reference solution. Take the reference solution and measure it 6 times in a row, and calculate the RSD value of the ratio of the peak area of ​​the target substance to the peak area of ​​the internal standard substance. The results are shown in the table below:

[0104] Table 10: Instrument precision results (ratio of target peak area to internal standard peak area)

[0105] frequency Peak area ratio 1 0.7343 2 0.7305 3 0.7268 4 0.7286 5 0.7236 6 0.7285 RSD(%) 0.49

[0106] Results: The RSD of the ratio of the peak area of ​​the target substance to the internal standard substance was 0.49%, and the instrument precision was good. 4.9 Summary of the methodological validation of non-deuterated TSL-0202 in TSL-0202

[0107] Table 11: Summary of Methodological Validation of Non-deuterated TSL-0202 in TSL-0202

[0108]

[0109]

[0110] There is no literature report on the determination of non-deuterated TSL-0202 content in TSL-0202, so the research on it is of great significance. 1 HNMR technology, with succinic anhydride as internal standard and deuterated chloroform as solvent, under ideal experimental conditions, select 1 HNMR spectrum at δ H The signal of 3.7427 is the quantitative peak of non-deuterated TSL-0202, located at δ H The signal of 3.0283 is the quantitative peak of the succinic anhydride internal standard, and the non-deuterated TSL-0202 content in the sample to be tested is measured to be 0.1727% (RSD=4.03%). The method established in the present invention is simple to operate, fast and accurate, and does not destroy the sample. It provides a new means for determining the impurity content in the research and development process of TSL-0202, and also provides an experimental basis for its quality control research. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 Non-deuterated TSL-0202 linear relationship curve

[0112] Figure 2 Non-deuterated TSL-0202, succinic anhydride, and a mixture of TSL-0202 1 HNMR spectrum DETAILED DESCRIPTION

[0113] Example 1

[0114] 1.1 Instrument

[0115] BRUKER AV-400 nuclear magnetic resonance instrument, XPE26 one-millionth electronic analytical balance.

[0116] 1.2 Samples and reagents

[0117] Non-deuterated TSL-0202 (homemade, content >98.7%); Succinic anhydride (Beijing Bailingwei Science and Technology Co., Ltd., content >99.42%); Deuterated chloroform (Beijing Bailingwei Science and Technology Co., Ltd., 99.8% deuterated, containing 0.03% (V / V) TMS); TSL-0202 API (homemade).

[0118] 1.3 Sample preparation

[0119] Take 2 mg of succinic anhydride, accurately weigh it, place it in a 2 ml volumetric flask, dissolve it with deuterated chloroform and dilute it to the mark as the internal standard solution stock solution. Accurately measure 0.25 ml of the internal standard solution stock solution, place it in a 5 ml volumetric flask, dilute it to the mark with deuterated chloroform as the internal standard solution. Take about 10 mg of TSL-0202 raw material, accurately weigh it, place it in a 1 ml volumetric flask, dissolve it with the internal standard solution and dilute it to the mark as the test solution.

[0120] Transfer the sample solution to the NMR tube, set the instrument parameters correctly, adjust the speed of the NMR tube so that the rotating edge does not interfere with the measured signal, and record the spectrum. Use the integration method to measure the peak area of ​​the characteristic peak and the peak area of ​​the internal standard peak under each sample, repeat the measurement 5 times, and take the average value. The content shall not exceed 2%.

[0121] 1.4 Determination of NMR experimental conditions

[0122] By analyzing the solvent, internal standard, and NMR experimental parameters, the NMR detection scheme was finally determined to use deuterated chloroform as the solvent and succinic anhydride as the internal standard. The test conditions were as follows: relaxation time d1 = 10 s; number of scans ns = 32 times; empty scan DS = 4 times; spectrum width SW = 20.0254 PM; sampling time AQ = 4.0894966 s; center frequency O1 = 2600.85 Hz; measured at room temperature 1 H NMR spectroscopy.

[0123] 1.5 Determination of non-deuterated TSL-0202 content

[0124] Transfer the sample solution to the NMR tube, set the instrument parameters correctly, adjust the speed of the NMR tube so that the rotating edge does not interfere with the measured signal, and record the spectrum. Use the integration method to measure the peak area of ​​the characteristic peak and the peak area of ​​the internal standard peak under each sample, repeat the measurement 5 times, and take the average value. The content of non-deuterated TSL-0202 in TSL-0202 is calculated as follows:

[0125]

[0126] Where W r is the weight of the internal standard;

[0127] A s and A rare the average peak areas of the characteristic peaks of the test sample and the internal standard peak, respectively;

[0128] E s and E r are the proton equivalent weight (mass) of the test sample and the internal standard, respectively (calculated by dividing the molecular weight by the number of protons in the characteristic peak). The results of the test sample are shown in Table 1.

[0129] Table 12 Determination results of non-deuterated TSL-0202 content

[0130]

[0131] Example 2

[0132] Take 2 mg of succinic anhydride, weigh it accurately, place it in a 2 ml volumetric flask, dissolve it with deuterated chloroform and dilute it to the mark as the internal standard solution stock solution. Accurately pipette 0.25 ml of the internal standard solution stock solution, place it in a 5 ml volumetric flask, dilute it to the mark with deuterated chloroform as the internal standard solution.

[0133] Take about 10 mg of TSL-0202 API from different batches, weigh accurately, place in a 1 ml volumetric bottle, dissolve with internal standard solution and dilute to scale as sample solution. Measure each solution 5 times and calculate the average value.

[0134] The sample solution was measured and the non-deuterated TSL-0202 content was calculated. The results are shown in the table below.

[0135] Table 13: Test results of samples from different batches

[0136] Sample batch Non-deuterium TSL-0202 content (%) 181104 0.1697％ 181105 0.1806％ 181106 0.1677％

[0137] Results: The TSL-0202-ND contents in the three batches of samples, 181104, 181105 and 181106, were 0.1697%, 0.1806% and 0.1677%, respectively, which were far less than the quantitative limit.

Claims

1. A method for determining the content of non-deuterated TSL-0202 in TSL-0202, the method comprising the following steps: Step 1, preparation of the test solution: first prepare an internal standard solution containing a deuterium compound, dissolve the test product TSL-0202 with the internal standard solution, and prepare a test solution of a determined concentration; The internal standard solution is prepared as follows: weigh an appropriate amount of succinic anhydride, dissolve it in deuterated chloroform, and prepare an internal standard solution; the concentration of the deuterium compound in the internal standard solution is 0.01 mg / mL-10 mg / mL; The test solution is prepared as follows: take an appropriate amount of TSL-0202 raw material, place it in a volumetric bottle, dissolve it with the internal standard solution and dilute it to the scale as the test solution; the concentration of TSL-0202 in the test solution is 0.01mg / mL-10mg / mL; Step 2, loading the sample solution into a nuclear magnetic resonance instrument for determining the type and number of hydrogen atoms in a molecule to obtain a nuclear magnetic resonance spectrum of the sample; The nuclear magnetic resonance instrument has a magnetic field frequency range of 300MHz-1.2GHz; The NMR conditions are: deuterated chloroform as solvent, succinic anhydride as internal standard, and the test conditions are as follows: relaxation time d1 ≥ 10s; scanning number ns ≥ 32 times; empty scan DS = 4 times; spectrum width SW = 18 ~ 22PM; sampling time AQ = 3.5 ~ 5.5s; center frequency O1 = 2500 ~ 2700HZ; 1H NMR spectrum was measured at room temperature; Step 3, according to the nuclear magnetic resonance spectrum, calculate the content of non-deuterated TSL-0202 in the test product TSL-0202.

2. The assay method according to claim 1, wherein The determination is to transfer the sample solution into a nuclear magnetic resonance tube, set the nuclear magnetic resonance condition parameters, adjust the speed of the nuclear magnetic resonance tube so that the rotating edge does not interfere with the signal to be measured, turn on the machine and record the spectrum, and measure 5 times.

3. The measuring method according to claim 1, wherein The calculation adopts the integration method to calculate the peak area of ​​each characteristic peak and the peak area of ​​the internal standard peak respectively, and calculates the average value of 5 measurements.

4. The determination method according to claim 1, the content of non-deuterium TSL-0202 in TSL-0202 is calculated according to the following formula: Where W r is the weight of the internal standard; A s and A r are the average peak areas of the characteristic peaks of the test sample and the internal standard peak, respectively; E s and E r are the proton equivalent weights of the test sample and the internal standard, respectively.

5. The assay method according to claim 1, wherein the steps are as follows: (1) Preparation of test solution: Accurately weigh an appropriate amount of succinic anhydride, dissolve it in deuterated chloroform, and prepare an internal standard solution with a concentration of about 0.05 mg / ml; accurately weigh 10 mg of TSL-0202 raw material, place it in a 1 ml volumetric flask, dissolve it in the internal standard solution and dilute to the scale as the test solution; (2) Determination of non-deuterated TSL-0202 content: transfer the sample solution into the NMR tube, set the instrument parameters correctly, adjust the NMR tube speed so that the rotating edge does not interfere with the measured signal, and record the spectrum; (3) The peak area of ​​the characteristic peak and the peak area of ​​the internal standard peak of each sample were measured by the integration method, and the measurement was repeated 5 times to take the average value. The content of non-deuterated TSL-0202 in TSL-0202 was calculated according to the following formula: Where W r is the weight of the internal standard; A s and A r are the average peak areas of the characteristic peaks of the test sample and the internal standard peak, respectively; E s and E r are the proton equivalent weights of the test sample and the internal standard, respectively; in, The NMR parameters are as follows: relaxation time d1=10s; number of scans ns=32 times; empty scan DS=4 times; spectrum width SW=20.0254PM; sampling time AQ=4.0894966s; center frequency O1=2600.85HZ; and the 1H NMR spectrum was measured at room temperature.

6. The assay method according to claim 1, wherein δ H 3.7427 (3H, S) is the quantitative peak of non-deuterated TSL-0202, δ H 3.0283 (4H, S) is the quantitative peak of succinic anhydride internal standard.

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