A method for detecting mutagenic impurities in anti-HIV drug intermediates

Abstract

The application belongs to the technical field of medical analysis, and discloses a method for detecting mutagenic impurities in anti-HIV drug intermediates. The chromatographic column used in the method has octadecylsilane bonded silica gel as a filler, and uses formic acid solution with a formic acid concentration of 0.2-0.4 wt.% and acetonitrile for gradient elution. The mutagenic impurities 030 in the anti-HIV drug intermediates are separated and detected at a column temperature of 28-32 DEG C. The method can detect trace amounts of mutagenic impurities 030 in the anti-HIV drug intermediates, and provides protection for the quality and safety of the anti-HIV drug during preparation. The detection method provided by the application is short in time, high in sensitivity, good in stability, and simple and feasible in operation.

Description

Technical Field

[0001] This invention relates to a method for detecting mutagenic impurities in anti-HIV drugs or their intermediates, belonging to the field of pharmaceutical technology. Background Technology

[0002] AIDS is a highly dangerous infectious disease caused by infection with the human immunodeficiency virus (HIV). To date, the anti-HIV drugs approved by the U.S. FDA are mainly divided into six categories: nucleoside / nucleotide reverse transcriptase inhibitors (N(t)RTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors (PI), fusion inhibitors, integrase strand transfer inhibitors (INSTI), and co-receptor inhibitors.

[0003] Asuptegravir is a novel oral anti-HIV drug with the chemical name: (4) R ,12 aS )- N- (2,4-Difluorophenyl)-7-hydroxy-4-methyl-6,8-dioxo-6,8,12,12 a- Tetrahydro-2 H 4 H- Spiro[cyclopropane-1,3-pyrido[1,2:4,5]pyrazine[2,1- b [1,3]oxazine]-9'-formamide inhibits the strand transfer activity of HIV-1 integrase (an HIV-1-encoding enzyme essential for viral replication), preventing linear DNA from integrating into the host genomic DNA and blocking HIV-1 proviral formation and viral replication.

[0004] (Asuptegravir structure).

[0005] CN116120342A discloses a method for preparing Asuptegravir, the reaction route of which is as follows: ; .

[0006] However, during the production process, it was discovered that raw material 1b.1 may remain in synthetic intermediate 1b.2; intermediate 1c (molecular formula: C) was synthesized from intermediate 1b.2. 23 H 23During the process of producing F2N3O5 or active pharmaceutical ingredient, residual raw material 1b.1 will continue to react and derive impurities such as 5-methoxy-6-(methoxycarbonyl)-4-oxo-1-(2-oxoethyl)-1,4,5,6-tetrahydropyridine-3-carboxylic acid, with the structural formula shown in formula (I).

[0007] Formula (I) Genotoxicity refers to the toxicity caused by any harmful change in genetic material, regardless of the mechanism that induces the change; it is also known as mutagenicity. Genotoxic impurities (GTIs) are impurities with genotoxic effects, including mutagenic impurities and other types of non-mutagenic impurities. They mainly originate from the production process of active pharmaceutical ingredients (APIs), such as starting materials, reaction intermediates, catalysts, reagents, and degradation products. Mutagenic impurities are impurities that can directly cause DNA damage and lead to mutations at low levels. The impurity shown in formula (I) has a mutagenic warning structure aldehyde group and needs to be controlled as a mutagenic impurity. Therefore, in the production process of Asuptegravir, detecting the content of the impurity shown in formula (I) in intermediates and strictly controlling the residual amount of the impurity shown in formula (I) within an appropriate and acceptable range is crucial for the quality control and safety of the drug.

[0008] A literature review has not yet revealed any reports of relevant methods, either domestically or internationally. Summary of the Invention

[0009] The purpose of this invention is to provide a method for detecting mutagenic impurity O30 in intermediates of anti-HIV drugs. This detection method is short in time, highly sensitive, stable, simple and feasible to operate, and provides assurance for the quality and safety of anti-HIV drugs.

[0010] To achieve the above objectives, the present invention provides a method for detecting mutagenic impurities in anti-HIV drug intermediates, wherein the mutagenic impurities include impurity 030 with the structure shown in formula (I); Formula (I); The method includes the following steps: using impurity 030 as a reference standard, liquid chromatography-mass spectrometry is used to detect whether the anti-HIV drug intermediate sample contains impurity 030 or to calculate the content of impurity 030 by external standard method; The detection conditions for the liquid chromatography-mass spectrometry method include: Column: Packed with octadecylsilane-bonded silica gel; Mobile phase A: Formic acid solution with a concentration of 0.2-0.4 wt.%; Mobile phase B: Acetonitrile; The elution method is gradient elution; Column temperature: 28-32℃.

[0011] In the course of research and development, to better control the product quality of anti-HIV drugs and their intermediates, and considering the warning structure of the aldehyde group in compound (I), the inventors of this invention commissioned a third party to analyze and evaluate compound (I). Specifically, in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH M7) guidelines, the toxicity assessment of the impurities was conducted using two complementary (Q)SAR prediction methods (one based on expert knowledge rules and the other based on statistics). The assessment was performed using the Derek Nexus & Sarah Nexus assessment software from Lhasa Limited. This method is a toxicity assessment method that uses computational methods to analyze, simulate, visualize, or predict the toxicity of chemicals. It has significant advantages in terms of time, cost, and animal welfare, and can be used to predict the results of bacterial mutagenicity tests. It has been widely accepted by the FDA, EMA, and CDE. The results of the two complementary prediction methods for compound (I) were positive (see results). Figure 1 Based on this, the inventors conducted a bacterial reverse mutation test (Ames test) on compound (I), which also showed that compound (I) was positive. That is, the evaluation results showed that compound (I) was a mutagenic impurity, and it is necessary to strictly monitor and determine its content in order to control the quality of anti-HIV drugs or their intermediates.

[0012] The high-performance liquid chromatography-mass spectrometry method provided by this invention can rapidly, accurately, and effectively separate and detect mutagenic impurity O30 in anti-HIV drug intermediates. This method has a short detection time, high sensitivity, good stability, and simple and feasible operation. It can be used as an effective method to detect whether the content of impurity O30 in anti-HIV drug intermediates meets the quality standards, which is conducive to improving the quality of anti-HIV drug raw materials (especially Asuptegravir) and ensuring the safety of patients' medication.

[0013] In this invention, mutagenic impurities in anti-HIV drug intermediates are separated and detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), specifically through qualitative or quantitative detection. The qualitative detection process includes: using impurity 030 (as shown in formula (I)) as a reference standard, determining the corresponding peak time using the HPLC-MS / MS detection conditions provided by this invention (e.g., the peak time of impurity 030 is approximately 3.9 min), and then detecting whether a chromatographic peak appears in the anti-HIV drug intermediate sample at or near the aforementioned peak time using the same HPLC-MS / MS detection conditions to determine whether the anti-HIV drug intermediate sample contains impurity 030. Quantitative detection can employ the external standard method. The specific process of the external standard method includes: using impurity 030 as a reference standard, preparing a reference standard solution, preparing a test solution from the anti-HIV drug intermediate sample, detecting the impurity 030 using the HPLC-MS / MS detection conditions provided by this invention, recording the chromatograms, and calculating the corresponding concentration of impurity 030 for quantification.

[0014] In this invention, the intermediate sample of the anti-HIV drug can be prepared by referring to known processes. For example, the structure of the anti-HIV drug is shown in formula (III), and the structure of the intermediate is shown in formula (II). Specific preparation processes can be found in the method disclosed in CN116120342A. Formula (III) Equation (II).

[0015] In a preferred embodiment, the method further includes: mixing the anti-HIV drug intermediate sample and the reference standard with a diluent to prepare a test solution and a reference solution, respectively, and then performing detection by liquid chromatography-mass spectrometry.

[0016] More preferably, the diluent contains an ammonium acetate solution with a pH of 3-4 and acetonitrile, and the volume ratio of the ammonium acetate solution to acetonitrile is 1:0.8-1.2, specifically 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, or any value between the two aforementioned values.

[0017] In a preferred embodiment, the concentration of ammonium acetate in the ammonium acetate solution is 9-11 mM, specifically 9 mM, 9.2 mM, 9.4 mM, 9.6 mM, 9.8 mM, 10 mM, 10.2 mM, 10.4 mM, 10.6 mM, 10.8 mM, 11 mM, or any value between the two aforementioned values.

[0018] In a preferred embodiment, the content of the anti-HIV drug intermediate sample in the test solution is 8-12 mg / ml, specifically 8 mg / ml, 8.5 mg / ml, 9 mg / ml, 9.5 mg / ml, 10 mg / ml, 10.5 mg / ml, 11 mg / ml, 11.5 mg / ml, 12 mg / ml, or any value between the two aforementioned values; the content of impurity O30 in the reference solution is 60-80 ng / ml, specifically 60 ng / ml, 62 ng / ml, 64 ng / ml, 66 ng / ml, 68 ng / ml, 70 ng / ml, 72 ng / ml, 74 ng / ml, 76 ng / ml, 78 ng / ml, 80 ng / ml, or any value between the two aforementioned values.

[0019] In this invention, a solution of ammonium acetate solution and acetonitrile mixture with a specific content and ratio is used as a solvent to prepare a solution of anti-HIV drug intermediate sample with a specific content, which can effectively improve the accuracy and stability of the separation and detection of mutagenic impurity O30 in anti-HIV drug intermediate.

[0020] In a preferred embodiment, the chromatographic column has a length of 150 mm, an inner diameter of 4.6 mm, and a packing particle size of 3 μm. More preferably, the chromatographic column is a Phenomenex Gemini. ® NX-C18 110 Å column.

[0021] In a preferred embodiment, the liquid chromatography-mass spectrometry detection conditions further include: the total flow rate of the mobile phase is 1-1.5 ml / min, specifically 1 ml / min, 1.1 ml / min, 1.2 ml / min, 1.3 ml / min, 1.4 ml / min, 1.5 ml / min, or any value between the two aforementioned values; more preferably 1.2 ml / min.

[0022] As a preferred embodiment, the liquid chromatography-mass spectrometry detection conditions further include: the detection wavelength of the ultraviolet detector is 265-275nm, specifically 265nm, 267nm, 269nm, 270nm, 271nm, 273nm, 275nm, or any value between the two aforementioned values; more preferably 270nm.

[0023] In a preferred embodiment, the ion source of the MS detector in the liquid chromatography-mass spectrometry is an ESI source, and the scanning is performed in SIM-negative ion mode for a scanning time of 2 to 4.5 min.

[0024] In this invention, the formic acid concentration of mobile phase A can specifically be 0.2 wt.%, 0.25 wt.%, 0.3 wt.%, 0.35 wt.%, 0.4 wt.%, or any value between the aforementioned two values. As a preferred embodiment, mobile phase A is a formic acid solution with a formic acid concentration of 0.3 wt.%, and mobile phase B is acetonitrile.

[0025] As a preferred embodiment, the gradient elution procedure is as follows: .

[0026] More preferably, the gradient elution procedure is as follows: .

[0027] In this invention, the column temperature can be 28℃, 28.5℃, 29℃, 29.5℃, 30℃, 30.5℃, 31℃, 31.5℃, 32℃, or any value between the two aforementioned values.

[0028] As a preferred embodiment, the liquid chromatography-mass spectrometry detection conditions further include: the injection volume is 15-25 μl, specifically 15 μl, 16 μl, 17 μl, 18 μl, 19 μl, 20 μl, 21 μl, 22 μl, 23 μl, 24 μl, 25 μl, or any value between the two aforementioned values.

[0029] The liquid chromatography-mass spectrometry method provided by this invention can detect any anti-HIV drug intermediate that uses impurity 030 as an intermediate material or contains intermediate materials capable of generating impurity 030. As a preferred embodiment, the structure of the anti-HIV drug intermediate is shown in formula (II). Equation (II).

[0030] As a preferred embodiment, a method for determining mutagenic impurities in anti-HIV drug intermediates using liquid chromatography-mass spectrometry includes the following steps: (1) Prepare a test solution from an anti-HIV drug intermediate sample and prepare a reference solution from impurity 030. The test solution and the reference solution are the same. (2) Each solution in step (1) is tested under the detection conditions of the above liquid chromatography-mass spectrometry method, and the content of impurity 030 is calculated by external standard method.

[0031] As a relatively preferred embodiment of the present invention, the method for determining mutagenic impurities in anti-HIV drug intermediates using liquid chromatography-mass spectrometry includes: (1) An ammonium acetate solution (pH 3-4) mixed with acetonitrile at a volume ratio of 1:0.8-1.2 was used as a diluent; Reference solution: Take an appropriate amount of impurity 030 reference standard, add diluent to dissolve and dilute to prepare a solution containing approximately 60-80 ng of impurity 030 per ml; Test solution: Take the anti-HIV drug intermediate sample, add diluent to dissolve and dilute to prepare a solution containing about 8-12 mg of anti-HIV drug intermediate sample per 1 ml; (2) Detect each solution in step (1) according to the detection conditions of liquid chromatography-mass spectrometry as follows, record the chromatogram and mass chromatogram, and calculate the content of impurity 030 in the sample according to the external standard method; Column: Octadecylsilane-bonded silica gel (Phenomenex Gemini) ® (NX-C18 110Å, 4.6mm × 150mm, 3μm or equivalent column). Mobile phase A: Formic acid solution with a concentration of 0.2-0.4 wt.%; Mobile phase B: Acetonitrile; The gradient elution procedure is as follows: ; Column temperature: 28-32℃; Flow rate: 1-1.5 ml / min; Detection wavelength: 265-275nm (e.g., 270nm); Injection volume: 15-25µl; The MS detector uses an ESI source as its ion source and scans in SIM-negative ion mode, scanning ion peak 268 for 2–4.5 min.

[0032] Compared with the prior art, the present invention has the following significant advantages: The present invention provides a method for detecting mutagenic impurity O30 in anti-HIV drug intermediates. By specifically selecting and synergistically combining the chromatographic column, mobile phase type, elution mode, column temperature, and other conditions in high-performance liquid chromatography-mass spectrometry, it is possible to detect trace amounts of mutagenic impurity O30 in anti-HIV drug intermediates. Moreover, the method has high detection sensitivity and good stability, and can be used as an effective method to detect whether the content of impurity O30 in anti-HIV drug intermediates meets the quality standards. In particular, it provides assurance for the quality and safety of the anti-HIV drug Asuptegravir during its preparation. Attached Figure Description

[0033] Figure 1 The QSAR prediction results for impurity 030 are shown. Figure 2 This is a typical mass chromatogram of a blank solution; Figure 3 This is a typical mass chromatogram of the reference solution; Figure 4 This is a typical mass chromatogram of the test solution; Figure 5 This is a typical chromatogram of the test solution; Figure 6 This is a typical mass chromatogram of a solution at the limit of quantitation. Figure 7 Typical mass chromatogram of solution at the detection limit; Figure 8 Linear curve of impurity 030; Figure 9 The chromatogram of the stability of the reference solution at -0h is shown. Figure 10 The mass chromatogram of the stability of the spiked solution of the test sample at 0 h; Figure 11 The chromatogram shows the stability of the reference solution after 18 hours. Figure 12 The mass chromatogram of the spiked solution of the test sample under the detection conditions of Example 1; Figure 13 The mass chromatogram of the spiked solution of the test sample under the detection conditions of Example 1, with the column temperature adjusted to 28°C; Figure 14 The mass chromatogram of the spiked solution of the test sample at a column temperature adjusted to 32°C under the detection conditions of Example 1; Figure 15 Mass chromatogram of the spiked solution of the test sample after adjusting the initial ratio of the mobile phase to 93:7 under the detection conditions of Example 1; Figure 16 Mass chromatogram of the spiked solution of the test sample after adjusting the initial ratio of the mobile phase to 91:9 under the detection conditions of Example 1; Figure 17 The mass chromatogram of the spiked solution of the test sample after replacing the chromatographic column of the test conditions in Example 1 with another chromatographic column of the same brand and model; Figure 18 This is the mass chromatogram of the reference solution under the conditions of Comparative Example 1; Figure 19 The chromatogram of the spiked solution of the test sample under the conditions of Comparative Example 4 is shown. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] Experimental materials The reference standard of compound (I) (i.e., impurity 030) was provided by Jiangsu Aidi Pharmaceutical Group Co., Ltd., with batch number 1230066-47-01; The test sample used in the examples is intermediate 1c of the anti-HIV drug Asuptegravir. The structural formula of intermediate 1c is shown in formula (II). The sample of intermediate 1c was prepared according to the method of Example 1 in document CN116120342A.

[0036] In the following examples, RSD is an abbreviation for Relative Standard Deviation.

[0037] Example 1: Establishment of a method for determining mutagenic impurity O30 in intermediate 1c using liquid chromatography-mass spectrometry (LC-MS). Instrument and detection wavelength: High performance liquid chromatography-mass spectrometry, with an ultraviolet detector at a wavelength of 270 nm; Column: octadecylsilane-bonded silica gel as packing material; Phenomenex Gemini ® NX-C18 110 Å column, 150 mm long, 4.6 mm inner diameter, 3 μm packing particle size; Mobile phase: 0.3 wt.% formic acid solution (mobile phase A) - acetonitrile (mobile phase B); Flow rate: 1.2 ml / min; Column temperature and injection volume: Column temperature 30℃, injection volume 20μl; MS detector: ESI source, SIM mode (negative ion), scanning ion peak 268, scan time: 2~4.5 min; The elution method is gradient elution, and the gradient elution conditions are shown in Table 1: Table 1 Elution conditions

[0038] Accurately weigh impurity 030 reference standard, and prepare a solution containing 70 ng of impurity 030 in 1 ml using 10 mM ammonium acetate solution (pH 3.5)-acetonitrile (1:1) as solvent. Mix thoroughly and use as reference standard solution. Accurately weigh the sample of intermediate 1c shown in formula (II), and prepare a solution containing 10 mg of intermediate 1c in 1 ml using 10 mM ammonium acetate solution (pH 3.5) and acetonitrile (1:1) as the solvent. This solution is used as the test solution.

[0039] Specific experimental procedures: Inject 20 μl of the reference solution and the test solution into the high performance liquid chromatography-mass spectrometry instrument and perform detection under the above conditions. Record the chromatogram and mass chromatogram, and examine the peak time and separation of each component.

[0040] The content of impurity 030 in the test sample is calculated using the following formula:

[0041] in: F —Correction factor; A 对1 —Peak area of ​​reference solution 1; V 对1 —The dilution volume of reference solution 1, in ml; C 对 —Content of reference standard; m 对1 —The sample weight of reference standard 1, in mg; —The average correction factor for all 6 injections of reference solution 1; A 供 —Peak area of ​​impurity 030 in the test solution; V 供 —The dilution volume of the test solution, in ml; m 供 —Sample weight of the test sample, mg; m 对2 —The sample weight of reference standard 2, in mg; A 对2 —Peak area of ​​reference solution 2; C (ppm) — The content of impurity 030 in the test sample, in ppm.

[0042] Limits for mutagenic impurities: Asuptegravir, as shown in formula (III), is an anti-HIV drug intended for long-term use. The maximum daily dose is calculated based on 200 mg / day of free plasma (i.e., 210 mg / day of sodium salt). Potential mutagenic impurity: Impurity 030. The control limit (ppm) is calculated according to the strictest limit standard (TTC) for long-term use in ICH guidelines (1.5 mg / day):

[0043] From the perspective of drug safety, the limit for impurity 030 is initially set at 7 ppm. The intermediate 1c shown in formula (II) is an intermediate of Asuptegravir. Impurity 030 can be transferred to Asuptegravir from intermediate 1c. Therefore, the limit for controlling impurity 030 in intermediate 1c is also set at 7 ppm.

[0044] Example 2: Validation of the method for determining mutagenic impurities in the intermediate shown in formula (II) using liquid chromatography-mass spectrometry. This embodiment validates the method established in Example 1 for determining the mutagenic impurity 030 in intermediate 1c as shown in formula (II) using high performance liquid chromatography-mass spectrometry. The validation includes specificity and system suitability, limits of quantitation and detection, linearity and range, repeatability, accuracy, solution stability, and robustness, as detailed below: (1) Specificity and system applicability Take an appropriate amount of impurity 030 reference standard and prepare a solution containing 70 ng of impurity 030 in 1 ml using 10 mM ammonium acetate solution (pH 3.5)-acetonitrile (1:1) as solvent. Mix thoroughly and use as reference standard solution. Prepare two parallel solutions. Accurately weigh the intermediate sample shown in formula (II) and prepare a solution containing 10 mg of the intermediate shown in formula (II) in 1 ml using 10 mM ammonium acetate solution (pH 3.5) and acetonitrile (1:1) as the solvent. This solution is used as the test solution.

[0045] 20 μL each of the blank solution (solvent), reference solutions (STD 1, STD 2), and test solution were injected into the high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument for detection. Chromatograms and mass chromatograms were recorded. The peak times of each component, the injection precision and recovery rate of the reference solutions, and the separation of each component were investigated. The results are shown in Table 2, and typical chromatograms are shown in [Table 2]. Figures 2-5 The results showed that the blank solution and the test solution did not interfere with the mass spectrometry peak of impurity 030, the main peak of the test solution could be completely eluted, and the injection precision and recovery rate of the reference solution were good, meeting the validation requirements.

[0046] Table 2 Results of Specificity and System Applicability Assessment

[0047] (2) Limit of quantitation and limit of detection tests Dilute the reference solution 10 times using the dilution method to prepare the limit of quantitation (S / N) solution. Detect the limit of quantitation (S / N) of the reference solution. 3 10) Dilute the reference solution 20 times using the dilution method to prepare the detection limit solution. Detect the detection limit (S / N) of the reference standard. 3 3) See the atlas. Figures 6-7 The results are shown in Table 3.

[0048] Table 3 Results of the study on limit of quantitation and limit of detection

[0049] The experimental results show that, under the conditions of this method, the detection limit (0.0035 μg / ml) and quantitation limit (0.0070 μg / ml) of impurity 030 meet the requirements, and the concentration of impurity 030 at the quantitation limit is less than 10% of the control limit, which indicates that the conditions of this method can effectively detect impurity 030.

[0050] (3) Standard curve and linear range test Take an appropriate amount of impurity 030 reference standard and prepare a solution containing 0.7 mg of impurity 030 per ml using 10 mM ammonium acetate solution (pH 3.5)-acetonitrile (1:1) as the solvent. Mix thoroughly and use this solution as the reference standard stock solution. Dilute and bring the reference standard stock solution to volume according to Table 4, shake well, and prepare a linear solution.

[0051] Table 4. Preparation of linear solutions

[0052] Accurately measure 20 mL of each of the above linear solutions and inject them into the high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. Record the mass chromatograms, measure the peak areas, and perform linear regression with peak area as the ordinate and concentration as the abscissa. The results are shown in the figure. Figure 8 See Table 5. The results show that the peak area of ​​impurity 030 has a good linear correlation with the measured concentration in the concentration range of 0.0070~0.1405µg / ml.

[0053]

[0054] (4) Solution stability Take an appropriate amount of impurity 030 reference standard and prepare a solution containing 70 ng of impurity 030 in 1 ml using 10 mM ammonium acetate solution (pH 3.5) - acetonitrile (1:1) as the solvent. Mix thoroughly and use as the reference solution.

[0055] Take appropriate amounts of the intermediate sample shown in formula (II) and impurity 030 reference standard, and prepare a solution containing 10 mg of the intermediate shown in formula (II) and 70 ng of impurity 030 in 1 ml using a solvent. This solution is used as the spiking solution for the test sample. Inject the reference standard solution and the spiking solution for the test sample at room temperature (around 25℃) at 0, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, and 21 hours, respectively. Record the peak areas and calculate the ratio of the peak area of ​​impurity 030 at different time points to the peak area at 0 hours. The results are shown in Table 6, and the chromatogram is shown in […]. Figures 9-11 .

[0056] Table 6 Solution stability results

[0057] The results showed that when the reference solution was left at room temperature for 18 hours, the ratio of the peak area of ​​impurity 030 to the initial peak area was in the range of 80.0% to 120.0%.

[0058] (5) Repeatability Take an appropriate amount of impurity 030 reference standard and prepare a solution containing 0.7 mg of impurity 030 per ml using 10 mM ammonium acetate solution (pH 3.5) - acetonitrile (1:1) as the solvent. Mix thoroughly and use as the reference standard stock solution.

[0059] Accurately weigh 200 mg of intermediate 1c as shown in formula (II), place it in a 20 ml volumetric flask, accurately measure 2 ml of the reference stock solution, dissolve and dilute to the mark with solvent, shake well, and use as a repeatable solution. Prepare 6 parallel aliquots (freshly prepared before use).

[0060] Take a repeat solution and measure 6 solutions according to the method in Example 1. The results are shown in Table 7.

[0061] Table 7 Repeatability test results

[0062] Results: The repeatability of this method for determining samples was good.

[0063] (6) Accuracy test Take an appropriate amount of impurity 030 reference standard and prepare a solution containing 0.7 mg of impurity 030 per ml using 10 mM ammonium acetate solution (pH 3.5) - acetonitrile (1:1) as the solvent. Mix thoroughly and use as the reference standard stock solution.

[0064] Take 200 mg of intermediate 1c as shown in formula (II) accurately, place it in a 20 ml volumetric flask, and accurately measure 1 ml (3.5 ppm concentration), 2 ml (7 ppm concentration), and 3 ml (10.5 ppm concentration) of the reference stock solution respectively. Dissolve and dilute to the mark with solvent, shake well, and prepare 50%, 100%, and 150% accuracy solutions. Prepare 3 parallel portions of each accuracy solution (freshly prepared before use).

[0065] Accurately weigh 200 mg of intermediate 1c as shown in formula (II), place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and use it as the test solution.

[0066] Accurately pipette 20 mL of the reference solution, test solution, and accuracy solution into the high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument, and perform detection according to the conditions in Example 1. Record the mass chromatograms. The results are shown in Table 8.

[0067] Table 8 Accuracy Measurement Results

[0068] Calculation formula: Background = Sample weight of intermediate 1c as shown in formula (II) × Average content of impurities 030 in the test solution Recovery amount = Measured amount - Amount in the sample Recovery rate = (Recovered amount / Added amount) × 100%; Conclusion: The spiked recoveries of impurity 030 ranged from 97.6% to 112.2%, with an average of 103.4%, which met the validation requirements.

[0069] (7) Durability The spiked solution of the test sample was prepared according to the repeatability solution (freshly prepared before use) for robustness testing. Based on the detection conditions in Example 1, only single factors such as different column temperatures, different initial mobile phase ratios, and different batches of the same type of column were adjusted. The detection results are shown in Table 9, and the chromatograms are shown in […]. Figures 12-17 .

[0070] Table 9 Durability test results

[0071] The results showed that minor variations in the chromatographic conditions of this method had no significant effect on the determination of impurity 030 content.

[0072] Example 3: Application of the liquid chromatography-mass spectrometry method for determining mutagenic impurity O30 in intermediate 1c sample as shown in formula (II) Prepare the impurity 030 reference solution using the method described in Example 1; According to Example 1 in document CN116120342A, three batches of intermediate 1c as shown in formula (II) were prepared. The three batches of intermediate 1c samples were accurately weighed and prepared into a solution containing 10 mg of intermediate 1c per ml using 10 mM ammonium acetate solution (pH 3.5)-acetonitrile (1:1) as solvent. This solution was used as the test solution (freshly prepared before use).

[0073] The test was performed under the conditions described in Example 1.

[0074] Specific experimental procedures: 20 μl of the reference solution and the test solution were injected into the high performance liquid chromatography-mass spectrometry instrument for detection. The mass chromatograms were recorded and the results were calculated. The results are shown in Table 10.

[0075] Table 10 Detection results of impurity 030

[0076] The results showed that the content of the mutagenic impurity 030 in the three batches of samples all met the requirements.

[0077] Comparative Example 1 The same detection method and solution preparation process as in Example 1 were used, except that the SIM parameter in the MS detector was replaced with SIM mode (positive ion), and the ion peak at 288 was scanned. The results are as follows: Figure 18 As shown.

[0078] It can be seen that the molecular weight of the positive ion extracted from impurity 030 in SIM mode is 288. Although the response is strong, the baseline near the peak of impurity 030 is raised, which interferes with the detection of impurity 030.

[0079] Comparative Example 2 The same detection method and solution preparation process as in Example 1 were used, except that the solvent in Example 1, 10 mM ammonium acetate solution (pH 3.5) - acetonitrile (1:1), was replaced with acetonitrile-water-acetic acid (500:500:5) to prepare a reference solution for impurity 030. The stability of the reference solution at room temperature (around 25°C) was investigated. Samples were injected at 0, 1.5, 3, 4.8, 6, and 8 hours, and the peak areas were recorded. The ratio of the peak area of ​​impurity 030 at different time points to the peak area at 0 hours was calculated. The results are shown in Table 11. It can be seen that the reference solution prepared with acetonitrile-water-acetic acid (500:500:5) is stable at room temperature for 6 hours, and the ratio of the peak area of ​​impurity 030 to the initial peak area is in the range of 80.0% to 120.0%.

[0080] In Example 1, a 10 mM ammonium acetate solution (pH 3.5)-acetonitrile (1:1) was selected as the solvent. The reference solution was stable at room temperature for 18 hours (Table 6).

[0081] Table 11 Results of solution stability of the reference solution

[0082] Comparative Example 3 The same detection method and solution preparation process as in Example 1 were used, except that the injection volume was increased from 20 ml to 50 ml in Example 1. The impurity 030 reference solution was injected to detect the impurity. The mass chromatogram showed that the peak of impurity 030 was split due to the solvent effect.

[0083] Comparative Example 4 The same detection method and solution preparation process as in Example 1 and Example 2 were used, except that the flow rate was reduced from 1.2 ml / min to 1.0 ml / min in Example 1. The spiked solution was then injected for analysis, and it was found that the elution effect of impurity 030 was unsatisfactory. Further adjustments to the elution program according to Table 12 resulted in effective elution, and the chromatogram was recorded (see Table 12). Figure 19 The intermediate 1c peak was completely eluted within about 11 minutes, but the total running time was extended to 18 minutes.

[0084] Table 12 Elution Procedure

[0085] In summary, the method for determining mutagenic impurities in the intermediate shown in formula (II) using high performance liquid chromatography-mass spectrometry provided by this invention can rapidly, accurately, and effectively separate and detect mutagenic impurity 030 in anti-HIV drug intermediates, which is beneficial to improving the quality of anti-HIV drug raw materials and ensuring the safety of patients' medication.

Claims

1. A method for detecting mutagenic impurities in intermediates of anti-HIV drugs, characterized in that, The mutagenic impurities include impurity 030 with a structure as shown in formula (I); Equation (I); The method includes the following steps: using impurity 030 as a reference standard, liquid chromatography-mass spectrometry is used to detect whether the anti-HIV drug intermediate sample contains impurity 030 or to calculate the content of impurity 030 by external standard method; The detection conditions for the liquid chromatography-mass spectrometry method include: Column: Packed with octadecylsilane-bonded silica gel; Mobile phase A: Formic acid solution with a concentration of 0.2-0.4 wt.%; Mobile phase B: Acetonitrile; The elution method is gradient elution; Column temperature: 28-32℃.

2. The method according to claim 1, characterized in that, The method further includes: mixing the anti-HIV drug intermediate sample and the reference standard with a diluent to prepare a test solution and a reference solution, respectively, and then performing detection by liquid chromatography-mass spectrometry; Preferably, the diluent contains an ammonium acetate solution with a pH of 3-4 and acetonitrile, and the volume ratio of the ammonium acetate solution to acetonitrile is 1:0.8-1.

2.

3. The method according to claim 2, characterized in that, The concentration of ammonium acetate in the ammonium acetate solution is 9-11 mM.

4. The method according to claim 2, characterized in that, The content of the anti-HIV drug intermediate sample in the test solution is 8-12 mg / ml, and the content of impurity O30 in the reference solution is 60-80 ng / ml.

5. The method according to claim 1, characterized in that, The chromatographic column has a length of 150 mm, an inner diameter of 4.6 mm, and a packing particle size of 3 μm. Preferably, the chromatographic column is a Phenomenex Gemini. ® NX-C18 110 Å column.

6. The method according to claim 1, characterized in that, The gradient elution procedure is as follows: ； Preferably, the gradient elution procedure is as follows: 。 7. The method according to claim 1, characterized in that, The detection conditions for the liquid chromatography-mass spectrometry method also include: a total flow rate of 1-1.5 ml / min for the mobile phase; and a detection wavelength of 265-275 nm for the ultraviolet detector.

8. The method according to claim 1, characterized in that, In the liquid chromatography-mass spectrometry method, the ion source of the MS detector is an ESI source, and the scanning mode is SIM-negative ion mode, with a scanning time of 2~4.5 min.

9. The method according to claim 1, characterized in that, The injection volume for the liquid chromatography-mass spectrometry method is 15-25 μl.

10. The method according to any one of claims 1-9, characterized in that, The structure of the anti-HIV drug intermediate is shown in formula (II). Equation (II).

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