A method for detecting related substances in NP-01

Abstract

The present invention relates to the technical field of drug analysis, and specifically discloses a method for detecting related substances in NP-01. The detection method comprises the following steps: preparing a test solution and a system suitability solution: using liquid chromatography to detect the test solution and the system suitability solution, and the chromatographic conditions of the liquid chromatography are: using an Agilent ZORBAX Bonus-RP chromatographic column, with a trifluoroacetic acid aqueous solution having a volume concentration of 0.08% to 0.1% as mobile phase A, with methanol as mobile phase B, gradient elution, and a detection wavelength of 243nm to 247nm. The detection method provided by the present invention has fast analysis time, can quickly and accurately detect impurities in NP-01 raw materials, the whole operation process is reliable and controllable, suitable for practical application and promotion, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting related substances in NP-01. Background Art

[0002] The structural formula of compound NP-01 is shown in formula (I), and it exhibits excellent anti-tumor activity in various tumor animal models such as gastric cancer, liver cancer, lung cancer, and prostate cancer.

[0003]

[0004] In the process of synthesizing NP-01 API by existing technology, byproducts, starting materials or intermediates may be introduced, such as 11 impurities, such as BZP00177, BZP00181, BZP00176, BZP00178, BZP00129, BZP00128, BZP00155, BZP00171, BZP00170, BZP00127 and BZP00268, and there is no relevant literature reporting the detection method of these 11 impurities. Therefore, developing a method for detecting related substances in NP-01 API is of great significance to improve drug safety and quality control of NP-01 API. Summary of the invention

[0005] In view of this, the present application provides a method for detecting related substances in NP-01, which has strong specificity and excellent sensitivity, accuracy and linearity, and can meet the detection requirements of 11 impurities in NP-01 raw materials, including impurity BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127 and impurity BZP00268.

[0006] The structures of the above 11 impurities are as follows:

[0007]

[0008]

[0009] In order to achieve the above-mentioned object of the invention, the embodiment of the present invention adopts the following technical solution:

[0010] A method for detecting related substances in NP-01, the detection method comprising the following steps:

[0011] Step 1: Prepare the test solution and system suitability solution:

[0012] Preparation of test solution: Take NP-01 sample and prepare test solution with solvent;

[0013] System suitability solution: take impurity BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127, impurity BZP00268 and NP-01 reference substance, and prepare a system suitability solution with solvent;

[0014] Step 2: Using liquid chromatography to detect the test solution and the system suitability solution, the chromatographic conditions of the liquid chromatography are:

[0015] An Agilent ZORBAX Bonus-RP chromatographic column was used, with a trifluoroacetic acid aqueous solution with a volume concentration of 0.08% to 0.1% as mobile phase A, methanol as mobile phase B, gradient elution, and a detection wavelength of 243 nm to 247 nm.

[0016] Compared with the prior art, the detection method of the related substances in NP-01 provided in this application has the following advantages:

[0017] The detection method of related substances in NP-01 provided in the present application realizes the quantitative analysis of 11 impurities in the raw material drug, including impurity BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127 and impurity BZP00268. The method has strong specificity and high sensitivity, and meets the detection requirements of 11 impurities in the NP-01 raw material drug. Moreover, it has good linear relationship and high repeatability, and the correlation coefficients within the linear range of the standard curve are all greater than 0.99. In addition, the detection method also has a fast analysis time, and can quickly and accurately detect impurities in the NP-01 raw material drug. The entire operation process is reliable and controllable, suitable for practical application and promotion, and has broad application prospects.

[0018] Optionally, the gradient elution procedure is:

[0019] 0min→15min, 82%~78%→60% mobile phase A, 18%~22%→40% mobile phase B;

[0020] 15min→25min, 60%→50% mobile phase A, 40%→50% mobile phase B;

[0021] 25 min → 35 min, 50% → 45% mobile phase A, 50% → 55% mobile phase B;

[0022] 35 min → 45 min, 45% → 10% mobile phase A, 55% → 90% mobile phase B;

[0023] 45 min → 49 min, 10% mobile phase A, 90% mobile phase B;

[0024] 49 min → 49.1 min, 10% → 80% mobile phase A, 90% → 20% mobile phase B;

[0025] 49.1min→55min, 80% mobile phase A, 20% mobile phase B.

[0026] Further optionally, the procedure of the gradient elution is:

[0027] 0 min → 15 min, 80% → 60% mobile phase A, 20% → 40% mobile phase B;

[0028] 15min→25min, 60%→50% mobile phase A, 40%→50% mobile phase B;

[0029] 25 min → 35 min, 50% → 45% mobile phase A, 50% → 55% mobile phase B;

[0030] 35 min → 45 min, 45% → 10% mobile phase A, 55% → 90% mobile phase B;

[0031] 45 min → 49 min, 10% mobile phase A, 90% mobile phase B;

[0032] 49 min → 49.1 min, 10% → 80% mobile phase A, 90% → 20% mobile phase B;

[0033] 49.1min→55min, 80% mobile phase A, 20% mobile phase B.

[0034] Gradient elution has a significant impact on the separation effect and peak shape of the detection target. Therefore, the preferred gradient elution sequence of the present application can separate 11 impurities from NP-01 with excellent separation effect, with a separation degree between impurities greater than 1.2 and a separation degree between the main peak and the adjacent impurity peak greater than 1.5.

[0035] Optionally, the mobile phase A is a trifluoroacetic acid aqueous solution with a volume concentration of 0.1%.

[0036] Optionally, the detection wavelength is 245 nm.

[0037] Optionally, the column temperature is 30°C to 40°C, and the flow rate is 1.0ml / min to 1.2ml / min.

[0038] Further optionally, the column temperature is 35° C. and the flow rate is 1.1 ml / min.

[0039] The optimal flow rate and column temperature can make the 11 impurities and NP-01 peak in a shorter time, thus reducing the analysis time, and have excellent separation effect and better peak shape.

[0040] Optionally, the chromatographic column has a specification of 4.6 mm×250 mm×5 μm.

[0041] Optionally, the solvent is a mixed solution of methanol and water in a volume ratio of 7:3.

[0042] The preferred solvent can extract the NP-01 bulk drug and the 11 impurities contained therein as much as possible, and has substantially no interference with the liquid chromatography detection, thereby ensuring that the liquid chromatography detection result provided by the present invention is more accurate.

[0043] Optionally, the injection volume is 10 μl. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 is a high performance liquid chromatogram of a blank solvent provided in Example 1 of the present invention;

[0046] Figure 2 is a high performance liquid chromatogram of the test solution provided in Example 1 of the present invention;

[0047] Figure 3 is a high performance liquid chromatogram of the control solution provided in Example 1 of the present invention;

[0048] Figure 4 is a high performance liquid chromatogram of the system suitability solution provided in Example 1 of the present invention

[0049] Figure 5 is a linear relationship diagram of impurity BZP00177 provided in Example 2 of the present invention;

[0050] Figure 6 is a linear relationship diagram of impurity BZP00181 provided in Example 2 of the present invention;

[0051] Figure 7 is a linear relationship diagram of impurity BZP00176 provided in Example 2 of the present invention;

[0052] Figure 8 is a linear relationship diagram of impurity BZP00178 provided in Example 2 of the present invention;

[0053] Fig. 9 is a linear relationship diagram of impurity BZP00129 provided in Example 2 of the present invention;

[0054] Fig.10 is a linear relationship diagram of impurity BZP00128 provided in Example 2 of the present invention;

[0055] Fig.11 is a linear relationship diagram of impurity BZP00155 provided in Example 2 of the present invention;

[0056] Fig.12 is a linear relationship diagram of impurity BZP00171 provided in Example 2 of the present invention;

[0057] Fig.13 is a linear relationship diagram of impurity BZP00170 provided in Example 2 of the present invention;

[0058] Fig.14 is a linear relationship diagram of impurity BZP00127 provided in Example 2 of the present invention;

[0059] Fig.15 is a linear relationship diagram of the main component NP-01 provided in Example 2 of the present invention;

[0060] Fig.16 It is a linear relationship diagram of impurity BZP00268 provided in Example 2 of the present invention. DETAILED DESCRIPTION

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

[0062] Example 1

[0063] This embodiment provides a method for detecting related substances in NP-01, and the detection method comprises the following steps:

[0064] Step 1: Prepare blank solvent, test solution, control solution and system suitability solution:

[0065] Blank solvent: a mixed solution of methanol and water in a volume ratio of 7:3;

[0066] Test solution: Take 15 mg of NP-01 sample, weigh accurately, put into a 50 ml volumetric flask, add solvent to dissolve by ultrasonication and make up to the mark, shake well;

[0067] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well;

[0068] Mixed impurity stock solution: take impurity BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127 and impurity BZP00268, and prepare a mixed impurity stock solution with a solvent in which the concentration of each impurity is 3 μg / ml;

[0069] System adaptability solution: Take about 15 mg of NP-01 reference substance, weigh it accurately, put it in a 50 ml volumetric flask, add appropriate amount of solvent and sonicate to dissolve it, then accurately add 5 ml of the mixed impurity stock solution, and quantitatively dilute it with solvent to make a mixed solution containing about 0.3 mg of NP-01 and 0.3 μg of each impurity per 1 ml.

[0070] The above solvents are all mixed solutions of methanol and water in a volume ratio of 7:3.

[0071] Step 2: Use liquid chromatography to detect the blank solvent, test solution, control solution and system suitability solution, and record the spectra. The chromatograms are as follows: Figure 1 to Figure 4 As shown, the chromatographic conditions of the liquid chromatography are:

[0072] Agilent ZORBAX Bonus-RP 4.6mm×250mm×5μm chromatographic column was used, trifluoroacetic acid aqueous solution with a volume concentration of 0.1% was used as mobile phase A, methanol was used as mobile phase B, gradient elution, detection wavelength was 245nm, flow rate was 1.1ml / min, column temperature was 35°C, and injection volume was 10μl; wherein the procedure of the above gradient elution was as follows:

[0073] 0 min → 15 min, 80% → 60% mobile phase A, 20% → 40% mobile phase B;

[0074] 15min→25min, 60%→50% mobile phase A, 40%→50% mobile phase B;

[0075] 25 min → 35 min, 50% → 45% mobile phase A, 50% → 55% mobile phase B;

[0076] 35 min → 45 min, 45% → 10% mobile phase A, 55% → 90% mobile phase B;

[0077] 45 min → 49 min, 10% mobile phase A, 90% mobile phase B;

[0078] 49 min → 49.1 min, 10% → 80% mobile phase A, 90% → 20% mobile phase B;

[0079] 49.1min→55min, 80% mobile phase A, 20% mobile phase B.

[0080] from Figure 2 It can be seen that the separation of impurities BZP00176, impurity BZP00155, impurity BZP00268 and main component NP-01 is good, and 4 unknown peaks appear. Impurities BZP00177, impurity BZP00181, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00171, impurity BZP00170 and impurity BZP00127 were not detected. Among them, the chromatographic peak of impurity BZP00176 appeared at the retention time of 12.964min, the chromatographic peak of impurity BZP00155 appeared at the retention time of 22.962min, the chromatographic peak of main component NP-01 appeared at the retention time of 32.120min, and the chromatographic peak of impurity BZP00268 appeared at the retention time of 34.271min.

[0081] from Figure 4It can be seen that the chromatographic peaks of NP-01 raw material (main component NP-01) and impurities BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127, and impurity BZP00268 are well separated, among which the chromatographic peak of impurity BZP00177 appears at a retention time of 10.006 min, the chromatographic peak of impurity BZP00181 appears at a retention time of 11.624 min, the chromatographic peak of impurity BZP00176 appears at a retention time of 13.026 min, and the chromatographic peak of impurity BZP00177 appears at a retention time of 13.899 min. The chromatographic peak of impurity BZP00178 appeared, the chromatographic peak of impurity BZP00129 appeared at the retention time of 16.091min, the chromatographic peak of impurity BZP00128 appeared at the retention time of 20.582min, the chromatographic peak of impurity BZP00155 appeared at the retention time of 23.099min, the chromatographic peak of impurity BZP00171 appeared at the retention time of 25.001min, the chromatographic peak of impurity BZP00170 appeared at the retention time of 29.104min, the chromatographic peak of impurity BZP00127 appeared at the retention time of 30.225min, the chromatographic peak of main component NP-01 appeared at the retention time of 32.399min, and the chromatographic peak of impurity BZP00268 appeared at the retention time of 34.592min.

[0082] from Figure 4 It can also be seen that the separation between the NP-01 peak and the adjacent impurity peaks is greater than 1.5.

[0083] Example 2 Linear

[0084] 5.605 g of impurity BZP00177 with a content of 98.90% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 1 with a concentration of 110.8669 μg / ml.

[0085] 5.225 g of impurity BZP00181 with a content of 90.30% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 2 with a concentration of 94.3635 μg / ml.

[0086] 5.27 g of impurity BZP00176 with a content of 85.10% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 3 with a concentration of 89.6954 μg / ml.

[0087] 5.420 g of impurity BZP00178 with a content of 88.00% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 4 with a concentration of 95.3920 μg / ml.

[0088] 5.380 g of impurity BZP00129 with a content of 98.10% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 5 with a concentration of 105.5556 μg / ml.

[0089] 5.420 g of impurity BZP00128 with a content of 98.16% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 6 with a concentration of 106.4054 μg / ml.

[0090] 5.680 g of impurity BZP00155 with a content of 98.30% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 7 with a concentration of 111.6688 μg / ml.

[0091] 5.505 g of impurity BZP00171 with a content of 97.80% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 8 with a concentration of 107.6778 μg / ml.

[0092] 5.280 g of impurity BZP00170 with a content of 95.20% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 9 with a concentration of 100.5312 μg / ml.

[0093] 5.325 g of impurity BZP00127 with a content of 97.60% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 10 with a concentration of 103.9440 μg / ml.

[0094] 10.240 g of the main component NP-01 with a content of 86.16% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 11 with a concentration of 88.2278 μg / ml.

[0095] 5.315 g of impurity BZP00268 with a content of 84.8% was accurately weighed and dissolved in a mixed solution of methanol and water with a volume ratio of 7:3 to prepare a stock solution 12 with a concentration of 90.1424 μg / ml.

[0096] Accurately weigh several of the above stock solutions 1 to 12 to prepare mixed stock solutions, wherein the mixed stock solutions contain BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127, main component NP-01 and impurity The concentrations of BZP00268 were 5.5433μg / ml, 4.7182μg / ml, 4.4848μg / ml, 4.7696μg / ml, 5.2778μg / ml, 5.3203μg / ml, 5.5834μg / ml, 5.3839μg / ml, 5.0266μg / ml, 5.1972μg / ml, 4.4114μg / ml and 4.5071μg / ml respectively.

[0097] The mixed stock solution was diluted with a mixed solution of methanol and water in a volume ratio of 7:3 to prepare a series of concentration solutions, and detected by liquid chromatography. The liquid chromatography conditions were as shown in Example 1. The spectrum was recorded, and the concentration (μg / ml) was used as the abscissa and the peak area was used as the ordinate to draw a standard curve, and the regression equation was calculated. The results are shown in Tables 1 and Figures 5 to 16. From the results, it can be seen that the impurity BZP00177 has a good linear relationship in the concentration range of 0.0399μg / ml to 1.6630μg / ml; the impurity BZP00181 has a good linear relationship in the concentration range of 0.1415μg / ml to 1.4155μg / ml; the impurity BZP00176 has a good linear relationship in the concentration range of 0.0323μg / ml to 1.3454μg / ml; the impurity BZP00178 has a good linear relationship in the concentration range of 0.0343μg / ml to 1.4309μg / ml; the impurity BZP00129 has a good linear relationship in the concentration range of 0.0317μg / ml to 1.5833μg / ml; the impurity BZP00128 has a good linear relationship in the concentration range of 0.0511μg / ml to 1.5961μg / ml. The linear relationship is good; the impurity BZP00155 has a good linear relationship in the concentration range of 0.1675μg / ml to 1.6750μg / ml; the impurity BZP00171 has a good linear relationship in the concentration range of 0.1615μg / ml to 1.6152μg / ml; the impurity BZP00170 has a good linear relationship in the concentration range of 0.1508μg / ml to 1.5080μg / ml; the impurity BZP00127 has a good linear relationship in the concentration range of 0.1559g / ml to 1.5592μg / ml; the main component NP-01 has a good linear relationship in the concentration range of 0.0926g / ml to 1.3234μg / ml; the impurity BZP00268 has a good linear relationship in the concentration range of 0.1352g / ml to 1.3521μg / ml.

[0098] Table 1 Linearity test results

[0099]

[0100]

[0101]

[0102] Example 3 Detection Limit and Quantification Limit

[0103] Detection limit: The stock solutions 1 to 12 prepared in Example 2 were quantitatively diluted step by step with a mixed solution of methanol and water in a volume ratio of 7:3, and then detected by liquid chromatography. The specific conditions of the liquid chromatography were as described in Example 1. The spectrum was recorded and the detection limit was obtained at a signal-to-noise ratio of not less than 3:1.

[0104] Limit of quantification: The stock solutions 1 to 12 prepared in Example 2 were quantitatively diluted step by step with a mixed solution of methanol and water in a volume ratio of 7:3, and then detected by liquid chromatography. The specific conditions of the liquid chromatography were as described in Example 1. The spectrum was recorded, and the signal-to-noise ratio was not less than 10:1 as the limit of quantification.

[0105] The results of the detection limits and quantification limits of impurity BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127, impurity BZP00268 and main component NP-01 are shown in Table 2.

[0106] Table 2 Results of detection limit and quantification limit

[0107]

[0108] Example 4 Repeatability

[0109] Take the same batch of samples, prepare 6 test solutions according to the preparation method of the test solution in Example 1, and use liquid chromatography for detection. The specific conditions of liquid chromatography are as described in Example 1, and the results are shown in Table 3. As can be seen from the table, the RSD% of the 6 results of impurity BZP00176, impurity BZP00155, impurity BZP00268 and main component NP-01 are all less than 5%, and impurity BZP00177, impurity BZP00181, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00171, impurity BZP00170 and impurity BZP00127 are not detected, indicating that the detection method provided in this application has good repeatability.

[0110] Table 3 Repeatability test results

[0111] name 1 2 3 4 5 6 BZP00176(%) 0.01 0.01 0.01 0.01 0.01 0.01 BZP00155(%) 0.05 0.05 0.05 0.05 0.05 0.05 BZP00268(%) 0.04 0.04 0.04 0.04 0.04 0.04 Other largest impurities (%) 0.10 0.09 0.10 0.10 0.10 0.10 Total impurities% 0.24 0.24 0.25 0.24 0.25 0.25

[0112] Example 5 Intermediate Precision

[0113] Take the test solution prepared in Example 1 and detect it by liquid chromatography. The specific conditions of liquid chromatography are as described in Example 1. Different detection personnel use different chromatographic columns (replace different batches of chromatographic columns) to inject 6 times, each time 10 μL, and the test results are shown in Table 4. As can be seen from Table 4, the 6 results RSD% of impurity BZP00176, impurity BZP00155, impurity BZP00268 and main component NP-01 are all less than 5%, and impurity BZP00177, impurity BZP00181, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00171, impurity BZP00170 and impurity BZP00127 are not detected, indicating that the intermediate precision of the detection method provided by the present application is good.

[0114] Table 4 Intermediate precision test results

[0115]

[0116]

[0117] Example 6 Solution Stability

[0118] The test solution prepared in Example 1 was taken and placed for 0h, 1h, 2h, and 8h, respectively, and then detected by liquid chromatography. The specific conditions of liquid chromatography were as described in Example 1, and 10 μl was injected each time, and the peak area was recorded, and the area was normalized. The test results are shown in Table 5. As can be seen from Table 5, the test solution was placed at room temperature for 8 hours, and the contents of impurities BZP00176, impurity BZP00155 and impurity BZP00268 were stable, and impurities BZP00177, impurity BZP00181, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00171, impurity BZP00170 and impurity BZP00127 were not detected, indicating that the stability of the above test solutions was good.

[0119] Table 5 Stability test results

[0120]

[0121] Example 7 Recovery Test

[0122] The recovery rate was calculated according to the following formula:

[0123] Recovery rate (%) = (measured amount - original amount) / theoretical addition amount × 100%

[0124] Table 6 Recovery test results

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] As can be seen from Table 6, the recovery rates of impurity BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127 and impurity BZP00268 are all between 96.9% and 106.4%, and the RDS is less than 10%, which indicates that the detection method of the relevant substances in NP-01 provided in the present application has good accuracy.

[0131] Example 8

[0132] The system adaptability solution prepared in Example 1 was subjected to liquid chromatography analysis, the liquid chromatography conditions were fine-tuned, and the test results of the separation degree were recorded. From the results, it can be seen that the fine-tuning of the chromatographic conditions has no effect on the detection of 11 impurities and the main component NP-01, indicating that the detection method of the related substances in NP-01 provided by the present invention has good durability.

[0133] Fine-tuning 1. Column temperature adjustment: the column temperature is 30° C., and other conditions of liquid chromatography are the same as those in Example 1.

[0134] Fine-tuning 2. Column temperature adjustment: The column temperature was 40° C., and other conditions of liquid chromatography were the same as those in Example 1.

[0135] Fine-tuning 3. Wavelength adjustment: the wavelength is 243 nm, and other conditions of liquid chromatography are the same as those in Example 1.

[0136] Fine-tuning 4. Wavelength adjustment: the wavelength is 247 nm, and other conditions of liquid chromatography are the same as those in Example 1.

[0137] Fine-tuning 5, flow rate adjustment: the flow rate is 1.0 ml / min, and the other conditions of liquid chromatography are the same as those in Example 1.

[0138] Fine-tuning 6. Flow rate adjustment: the flow rate is 1.2 ml / min. Other conditions of liquid chromatography are the same as those in Example 1.

[0139] Fine-tuning 7. Mobile phase adjustment: Mobile phase A is a trifluoroacetic acid aqueous solution with a volume concentration of 0.08%. Other conditions of liquid chromatography are the same as those in Example 1.

[0140] Fine-tuning 8. Gradient elution adjustment: the initial volume ratio of mobile phase A to mobile phase B is 82:18, and the other conditions of liquid chromatography are the same as those in Example 1.

[0141] Fine-tuning 9. Gradient elution adjustment: the initial volume ratio of mobile phase A to mobile phase B is 78:22, and the other conditions of liquid chromatography are the same as those in Example 1.

[0142] Table 7 Separation fine-tuning test results

[0143]

[0144]

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances in NP-01, characterized in that: The detection method comprises the following steps: Step 1: Prepare the test solution and system suitability solution: Preparation of test solution: Take NP-01 sample and prepare test solution with solvent; System suitability solution: take impurity BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127, impurity BZP00268 and NP-01 reference substance, and prepare a system suitability solution with solvent; The related substances include impurity BZP00177, impurity BZP00181, impurity BZP00176, impurity BZP00178, impurity BZP00129, impurity BZP00128, impurity BZP00155, impurity BZP00171, impurity BZP00170, impurity BZP00127 and impurity BZP00268; Step 2: Using liquid chromatography to detect the test solution and the system suitability solution, the chromatographic conditions of the liquid chromatography are: An Agilent ZORBAX Bonus-RP chromatographic column was used, with a trifluoroacetic acid aqueous solution with a volume concentration of 0.08% to 0.1% as mobile phase A, methanol as mobile phase B, gradient elution, a detection wavelength of 243 nm to 247 nm, a column temperature of 30° C. to 40° C., and a flow rate of 1.0 ml / min to 1.2 ml / min; the specifications of the chromatographic column were 4.6 mm×250 mm×5 μm; The procedure of the gradient elution is: 0min→15min, 82%~78%→60% mobile phase A, 18%~22%→40% mobile phase B; 15min→25min, 60%→50% mobile phase A, 40%→50% mobile phase B; 25 min → 35 min, 50% → 45% mobile phase A, 50% → 55% mobile phase B; 35min→45min, 45%→10% mobile phase A, 55%→90% mobile phase B; 45 min → 49 min, 10% mobile phase A, 90% mobile phase B; 49 min → 49.1 min, 10% → 80% mobile phase A, 90% → 20% mobile phase B; 49.1min→55min, 80% mobile phase A, 20% mobile phase B.

2. The method for detecting related substances in NP-01 according to claim 1, characterized in that: The procedure of the gradient elution is: 0min→15min, 80%→60% mobile phase A, 20%→40% mobile phase B; 15min→25min, 60%→50% mobile phase A, 40%→50% mobile phase B; 25 min → 35 min, 50% → 45% mobile phase A, 50% → 55% mobile phase B; 35min→45min, 45%→10% mobile phase A, 55%→90% mobile phase B; 45 min → 49 min, 10% mobile phase A, 90% mobile phase B; 49 min → 49.1 min, 10% → 80% mobile phase A, 90% → 20% mobile phase B; 49.1min→55min, 80% mobile phase A, 20% mobile phase B.

3. The method for detecting related substances in NP-01 according to claim 1, characterized in that: The mobile phase A is a trifluoroacetic acid aqueous solution with a volume concentration of 0.1%.

4. The method for detecting related substances in NP-01 according to claim 1, characterized in that: The detection wavelength is 245 nm.

5. The method for detecting related substances in NP-01 according to claim 1, characterized in that: The column temperature was 35° C. and the flow rate was 1.1 ml / min.

6. The method for detecting related substances in NP-01 according to any one of claims 1 to 5, characterized in that: The solvent is a mixed solution of methanol and water in a volume ratio of 7:

3.

7. The method for detecting related substances in NP-01 according to any one of claims 1 to 5, characterized in that: The injection volume was 10 μl.

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