A method for detecting nitrosamine impurities in maraviroc dry suspension

Abstract

The application provides a detection method of nitrosamine impurities in maraviroc dry suspension, and belongs to the technical field of drug analysis, which comprises the following steps: detecting the nitrosamine impurities in the control sample solution, the test sample solution and the blank solution by using gas chromatography-tandem mass spectrometry, taking the ratio of the chromatographic peak area of the nitrosamine impurities to the chromatographic peak area of the corresponding internal standard substance as a quantitative parameter, and calculating the content of the nitrosamine impurities in the test sample solution by the internal standard method. According to the complex characteristics of the dry suspension matrix, a differential dissolution-special extraction-enhanced purification pretreatment system is constructed: the raw material is dissolved in DMSO, the remaining excipients are dispersed in water, special extraction is carried out by using methyl tert-butyl ether, and deep purification is carried out by combining sodium chloride salting-out and high-speed centrifugation. The problems that a plurality of nitrosamine impurities cannot be simultaneously detected due to the serious interference of the excipient matrix, the insufficient sensitivity and the lack of separation degree in the prior art are solved, and the specificity and accuracy of the detection are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for detecting nitrosamine impurities in mapasxavir dry suspension. Background Technology

[0002] Mapaxixavir is a novel Class 1 innovative anti-influenza drug that can effectively inhibit both influenza A and B viruses. Its pediatric formulation is a dry suspension, offering advantages such as pleasant taste, flexible dosage adjustment based on weight, and convenient administration. It is currently in clinical trials. The main active ingredient is ([[1'-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiazolin-11yl]-1',2',4',6'-tetrahydro-4',6'-dioxospiro[cyclopropane-1,3'-[3H]pyridino[1,2-b]pyridazine]-5'-yl]oxy]methyl carbonate), with the structure shown below. Figure 1 As shown, sodium nitrite is used in some steps of the manufacturing process of this active pharmaceutical ingredient. Given that sodium nitrite may react with secondary amine compounds present in the process, there is a potential risk of generating nitrosamine impurities. Specifically, residual tetrahydropyrrole and diisopropylamine in the process may react with nitrite to generate N-nitrosopyrrolidine (NPYR) and N-nitrosodiisopropylamine (NDIPA), respectively, which have high carcinogenic risks. Furthermore, dimethylamine introduced by the degradation of N,N-dimethylacetamide (DMAC) used in the process under specific conditions also poses a risk of reacting with nitrite to generate N-nitrosodimethylamine (NDMA). Nitrosamines are classified as carcinogens of very high concern by the International Agency for Research on Cancer (IARC), and according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) M7(R2) guidance, these impurities belong to the "cohort of concern" and require strict limit control in pharmaceuticals. Given that mapasxavir dry suspension is intended for pediatric use, its safety tolerances are more stringent, necessitating trace monitoring of any residual nitrosamine impurities. Therefore, establishing a detection method that can effectively separate NDMA, NDIPA, and NPYR, with sensitivity (QL down to the ng level) sufficient to meet the acceptable daily intake (AI) control threshold, high specificity, and ease of operation, is of significant practical importance and industrial value for ensuring the clinical safety and process quality evaluation of this drug.

[0003] Currently, the main methods for detecting nitrosamine impurities include gas chromatography-tandem mass spectrometry (GC-MS / MS) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). These methods have become the mainstream in the industry due to their high sensitivity and selectivity. However, existing nitrosamine detection methods are mostly applicable to other drugs or simple matrices such as tobacco, making them difficult to apply to mapasxavir dry suspension. This is because the excipient system of this suspension is complex (containing polymers, anhydrous lactose, mannitol, hydroxypropyl cellulose, sucralose, and other excipients), and its solubility characteristics and matrix effects are significant, easily causing serious interference to the extraction and separation of trace nitrosamines. Specifically, this manifests as low extraction recovery, peak tailing, and insufficient separation between the target analyte and interfering peaks, severely affecting quantitative accuracy. More importantly, the acceptable daily intake (AI) of nitrosamine impurities is typically at the nanogram level, placing extremely high demands on the sensitivity of detection methods. Conventional methods, due to poor matrix tolerance or insufficient sensitivity, are unable to meet the quality control release standards for this product.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting nitrosamine impurities in mapasixavir dry suspension, aiming to solve the technical problems in the prior art that make it impossible to simultaneously detect multiple nitrosamine impurities due to severe interference from excipient matrix, insufficient sensitivity and poor separation.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: This invention provides a method for detecting nitrosamine impurities in mapasixavir dry suspension, comprising using gas chromatography-tandem mass spectrometry (GC-MS / MS) to detect nitrosamine impurities in reference solution, test solution and blank solution, using the ratio of the chromatographic peak area of ​​nitrosamine impurity to the chromatographic peak area of ​​its corresponding internal standard as a quantitative parameter, and calculating the content of nitrosamine impurities in the test solution by the internal standard method; The reference solution, test solution, and blank solution all contain the same concentration of internal standard. The test solution was prepared by using mapasixavir dry suspension as the test sample, premixing with dimethyl sulfoxide, adding internal standard and mixing, dissolving in pure water, adding methyl tert-butyl ether and sodium chloride sequentially, vortexing and mixing, and then centrifuging to collect the supernatant and concentrating. The internal standard includes N-nitrosodimethylamine-D6 and / or N-nitrosodi-n-propylamine-D14; The reference standard is a nitrosamine impurity, which includes at least one of N-nitrosodimethylamine, N-nitrosodiisopropylamine, or N-nitrosopyrrolidine.

[0007] To address the complex characteristics of dry suspension matrices, a pretreatment system of "differentiated dissolution-dedicated extraction-enhanced purification" was constructed: first, the active pharmaceutical ingredient was dissolved in DMSO, then the remaining excipients were dispersed in water, followed by dedicated extraction with methyl tert-butyl ether, and deep purification was carried out in combination with sodium chloride salting-out and high-speed centrifugation, which completely solved the technical pain points of difficult dissolution of test samples and serious interference from excipient matrices.

[0008] Among them, the internal standard has similar physicochemical properties to nitrosamine impurities, which can effectively offset the errors in the sample pretreatment and detection process and ensure the accuracy of quantitative results.

[0009] Furthermore, in the chromatographic detection, the chromatographic column is a polar capillary column using polyethylene glycol as the stationary phase; the injection mode is splitless injection, and the injection volume is 2 μl. Increasing the injection volume from the conventional 1 μl to 2 μl forms a dual gain strategy of concentration + injection, which significantly improves the detection response value of nitrosamine impurities, while solving the problems of high low-point spiked recovery of NDIPA and insufficient low-concentration response value of NPYR.

[0010] The selection of a polar capillary column with a polyethylene glycol stationary phase should be based on the ability to effectively separate the three types of nitrosamine impurities from the matrix impurities. For example, an Agilent VF WAX ms (30m × 0.25mm × 1.00µm) can be selected.

[0011] Furthermore, the chromatographic conditions included: helium as the carrier gas (purity ≥99.999%), a flow rate of 0.95~1.05 mL / min to ensure good peak shape; an injection port temperature of 225~235℃; and a column temperature program of: initial temperature 40℃, hold for 1 min, then increase to 120℃ at a rate of 5℃ / min, hold for 2 min, and then increase to 240℃ at a rate of 10℃ / min, hold for 4 min.

[0012] Furthermore, the mass spectrometry conditions include: an electron impact ion source; an ion source temperature of 250°C; an interface temperature of 250°C; argon as the collision gas; and multiple reaction monitoring (MRM) mode. In practice, the electron energy and ion source voltage can be fine-tuned according to the instrument model to ensure optimal response intensity of the target compound's daughter ions; the peak cut-off time can be adjusted based on the actual peak elution time.

[0013] Furthermore, the quantitative ion pair for N-nitrosodimethylamine is 74 / 44, and the qualitative ion pair is 74 / 42; the quantitative ion pair for N-nitrosodimethylamine-D6 is 80 / 50, and the qualitative ion pair is 80 / 46; the quantitative ion pair for N-nitrosodiisopropylamine is 130 / 88, and the qualitative ion pair is 130 / 42; the quantitative ion pair for N-nitrosodi-n-propylamine-D14 is 144 / 126, and the qualitative ion pair is 78 / 50; and the quantitative ion pair for N-nitrosopyrrolidine is 100 / 70, and the qualitative ion pair is 100 / 55.

[0014] Furthermore, the internal standard method was used for quantification. The peak area ratio of each nitrosamine impurity in the reference solution to that of the internal standard was used as the quantitative parameter, and the content of each nitrosamine impurity in the test solution was calculated according to the standard curve method.

[0015] Furthermore, the detection method has a limit of quantification of ≤8.00 ng / g for N-nitrosodimethylamine, a limit of quantification of ≤2.20 ng / g for N-nitrosodiisopropylamine, and a limit of quantification of ≤141.52 ng / g for N-nitrosopyrrolidine. The linear correlation coefficient r ≥ 0.99 within the concentration range of 10–200%.

[0016] Furthermore, in the test sample solution, for every 1.2258 g to 1.4982 g of test sample, add 2.0 to 4.0 mL of dimethyl sulfoxide, 10.0 to 15.0 mL of pure water, 15.0 to 21.0 mL of methyl tert-butyl ether, and 1.0 to 3.0 g of sodium chloride; The supernatant collection includes collecting 2 / 3 of the total volume of the supernatant. The concentration includes concentrating to 1 / 10 of the volume.

[0017] Furthermore, the vortexing time is 30~60s; the concentration is carried out by nitrogen blowing, and the concentration temperature is ≤25℃; the centrifugation conditions include centrifugation at 8000rpm for ≥5min.

[0018] In the pretreatment of the test samples, the nitrogen blowing concentration temperature is controlled at room temperature to avoid the loss of nitrosamine impurities due to excessive temperature. At the same time, the centrifugation speed and vortex time parameters are clearly defined to ensure the consistency of each step of the operation. This method is suitable for the rapid detection of batch samples and provides reliable technical support for the quality control of mapasixavir dry suspension.

[0019] The pretreatment process requires no complex instruments or equipment, has low reagent costs, and is easy to implement, making it suitable for the routine testing conditions of pharmaceutical manufacturers and testing institutions. The testing process can meet the quality control requirements during the production and storage of mapasixavir dry suspension, providing strong assurance for the safety of clinical use of this drug.

[0020] Furthermore, the reference solution was prepared by diluting nitrosamine impurities with methyl tert-butyl ether and following the same method as the test solution. The reference solution contains solutions with N-nitrosodimethylamine concentrations of 36.32–145.34 ng / ml, N-nitrosodiisopropylamine concentrations of 10–40 ng / ml, and N-nitrosopyrrolidine concentrations of 642.5–2570 ng / ml. The blank solution is prepared without adding the test sample, following the same method as the test sample solution. The concentration of the internal standard in the reference solution, test solution, and blank solution is 25~100 ng / ml.

[0021] This invention provides a method for detecting nitrosamine impurities in mapasixavir dry suspension. Taking advantage of the fact that the excipients in mapasixavir dry suspension are readily soluble in water while the raw drug is essentially insoluble, a DMSO+water dissolution and dispersion scheme was selected through solubility testing. MTBE was used as the optimal extraction reagent, combined with sodium chloride to promote layering and high-speed centrifugation purification, ensuring complete sample dissolution and effectively eliminating matrix interference from excipients, thus improving the detection response value and resolution. Combined with internal standard quantification, the sensitivity, accuracy, and reproducibility of the detection are enhanced. This method can simultaneously and accurately detect three nitrosamine impurities—NDMA, NDIPA, and NPYR—in mapasixavir dry suspension. It boasts advantages such as high sensitivity, strong specificity, good accuracy, convenient operation, and high detection efficiency, meeting the quality control requirements of mapasixavir dry suspension and ensuring clinical medication safety. It effectively solves the technical problem in existing technologies where severe matrix interference from excipients, insufficient sensitivity, and poor resolution prevent the simultaneous detection of multiple nitrosamine impurities. This study fills the technical gap in the simultaneous detection of three nitrosamine impurities—NDMA, NDIPA, and NPYR—in mapasixavir dry suspension, providing important technical support for the formulation of quality standards for mapasixavir dry suspension, and also providing a reference for the detection of nitrosamine impurities in other dry suspension drugs. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a structural diagram of the active pharmaceutical ingredient; Figure 2 The linear response spectrum of N-nitrosodimethylamine (NDMA) provided in Experimental Example 1 of this invention; Figure 3 The linear response spectrum of N-nitrosodiisopropylamine (NDIPA) provided in Experimental Example 1 of this invention; Figure 4 The linear response spectrum of N-nitrosopyrrolidine (NPYR) provided in Experimental Example 1 of this invention; Figure 5 The chromatogram of the blank solution under the method for detecting nitrosamine impurities in the mapasixavir dry suspension provided in Experimental Example 1 of the present invention; Figure 6 This is a chromatogram of the reference solution under the method for detecting nitrosamine impurities in the mapasixavir dry suspension provided in Experimental Example 1 of the present invention. Figure 7 The chromatogram of the 100% spiked test solution is shown in the method for detecting nitrosamine impurities in the mapasixavir dry suspension provided in Experimental Example 1 of this invention. Figure 8 Chromatogram 1 is the lower detection limit solution of the macaveric acid dry suspension provided in Experimental Example 1 of this invention for detecting nitrosamine impurities. Figure 9 The chromatogram is the limit-of-quantity solution of the method for detecting nitrosamine impurities in the mapasixavir dry suspension provided in Experimental Example 1 of this invention. Detailed Implementation

[0024] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0025] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0028] Test sample: Mapaxixavir dry suspension (Health Yuan Pharmaceutical Group Co., Ltd.; batch number: 25095805).

[0029] Reference standards: N-nitrosodimethylamine reference standard (①TM standard: 1000 μg / ml; batch number: A25060251; ②Maclean: 99.857%; batch number: C17480558), N-nitrosodiisopropylamine reference standard TM standard: 100 μg / ml; batch number: 24080231), N-nitropyrrolidine reference standard (①TM standard: 100 μg / ml; batch number: A23070049; ②TM standard: 98.0%; batch number: 25060222).

[0030] Internal standard: A mixed standard solution of two nitrosamines in methanol (Tianjin Alta Technology Co., Ltd.: N-nitrosodimethylamine-D6 at 999.0 μg / ml, N-nitrosodi-n-propylamine-D14 at 999.1 μg / ml; batch number: S192039) Reagents: dichloromethane (Sigma-Aldrich: chromatographic grade; batch number: WXBF4778V), ultrapure water (≥18.2 MΩ•cm), dimethyl sulfoxide (AmPu: chromatographic grade; batch number: D802090), methyl tert-butyl ether (CNW: chromatographic grade; batch number: D708342), sodium chloride (Komeo: GR grade; batch number: 202111021).

[0031] Experimental Example 1 A method for detecting nitrosamine impurities in mapasxavir dry suspension, comprising the following steps: 1.1 Specific methods 1.1.1 Method conditions, as shown in Tables 1 to 3.

[0032] Table 1 Chromatographic conditions

[0033] Table 2 Mass Spectrometry Parameters

[0034] Table 3 MRM Parameters

[0035] Among them, N-nitrosodimethylamine was corrected using the internal standard N-nitrosodimethylamine-D6, and N-nitrosopyrrolidine and N-nitrosodiisopropylamine were corrected using the internal standard N-nitrosodi-n-propylamine-D14.

[0036] 1.1.2 Solution Preparation 1.1.2.1 Reagent blank: Take an appropriate amount of methyl tert-butyl ether into a 1.5 ml vial.

[0037] 1.1.2.2 N-Nitrosaminoglycan stock solution (1) N-nitrosodimethylamine stock solution 1: Weigh 45.69 mg of N-nitrosodimethylamine accurately, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with methyl tert-butyl ether, and shake well to obtain the solution. The concentration is 2281 μg / ml.

[0038] (2) N-nitrosodimethylamine stock solution 2: Accurately measure 438.4 μl of N-nitrosodimethylamine stock solution 1, place it in a 10 ml volumetric flask, dilute to the mark with methyl tert-butyl ether, and shake well to obtain the solution. The concentration is 100.0 μg / ml.

[0039] (3) N-nitrosodimethylamine stock solution 3: Accurately measure 400 μl of N-nitrosodimethylamine stock solution 2, place it in a 20 ml volumetric flask, dilute to the mark with methyl tert-butyl ether, and shake well to obtain the solution. The concentration is 2.000 μg / ml.

[0040] 1.1.2.3 N-Nitrosaminodiisopropylamine stock solution Accurately measure 100 μl of N-nitrosodiisopropylamine (100 μg / ml) in methanol, place it in a 5 ml volumetric flask, dilute to the mark with methyl tert-butyl ether, and shake well. The concentration is 2.000 μg / ml.

[0041] 1.1.2.4 N-Nitropyrrolidine Stock Solution (1) N-nitrosopyrrolidine stock solution 1: Weigh 28.18 mg of N-nitrosopyrrolidine accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with methyl tert-butyl ether, and shake well. The concentration is 2762 μg / ml.

[0042] (2) N-nitrosopyrrolidine stock solution 2: Accurately measure 144.8 μl of N-nitrosopyrrolidine stock solution 1, place it in a 20 ml volumetric flask, dilute to the mark with methyl tert-butyl ether, and shake well to obtain the solution. The concentration is 20.00 μg / ml.

[0043] 1.1.2.5 Internal Standard Mixed Stock Solution (1) Internal standard mixed standard stock solution 1: Accurately measure 50 μl of the mixed standard solution of two nitrosamines in methanol (1000 μg / ml), place it in a 5 ml volumetric flask, dilute to the mark with methyl tert-butyl ether, and shake well to obtain the solution. The concentration of NDMA-D6 is 9.990 μg / ml, and the concentration of NDPA-D14 is 9.991 μg / ml.

[0044] (2) Internal standard mixed standard stock solution 2: Accurately measure 1000 μl of internal standard mixed standard stock solution 1, place it in a 10 ml volumetric flask, dilute to the mark with methyl tert-butyl ether, and shake well to obtain the solution. The concentration of NDMA-D6 is 0.9990 μg / ml, and the concentration of NDPA-D14 is 0.9991 μg / ml.

[0045] (3) Internal standard mixed standard stock solution 3: Accurately measure 3 ml of internal standard mixed standard stock solution 2, place it in a 200 ml volumetric flask, dilute to the mark with methyl tert-butyl ether, and shake well to obtain the solution. The concentration of NDMA-D6 is 14.98 μg / ml, and the concentration of NDPA-D14 is 14.99 μg / ml.

[0046] 1.1.2.6 Reference Standard Mixed Stock Solution (1) Reference mixed stock solution 1: Accurately transfer 2.18 ml of N-nitrosodimethylamine stock solution 3, 0.6 ml of N-nitrosodiisopropylamine stock solution, and 3.856 ml of N-nitrosopyrrolidine stock solution 2 into the same 20 ml volumetric flask, add methyl tert-butyl ether to dilute to the mark, shake well, and the concentration after dilution is shown in Table 4.

[0047] (2) Reference mixed stock solution 2: Accurately transfer 10 ml of reference mixed stock solution 1 into a 100 ml volumetric flask, add methyl tert-butyl ether to dilute to the mark, shake well, and the concentration after dilution is shown in Table 4.

[0048] Table 4 Concentration of the mixed stock solution of reference standards

[0049] 1.1.2.7 Blank solution (MB), limit of detection solution (DL), limit of quantitation solution (QL), standard solution (STD), and linear solutions (L1~L6) Accurately transfer 2 ml of dimethyl sulfoxide (DMSO), 5 ml of internal standard mixed stock solution 3, and an appropriate amount of reference standard mixed stock solution 2 into a 50 ml centrifuge tube. Vortex for 30 seconds. Accurately transfer 13 ml of ultrapure water, add an appropriate amount of methyl tert-butyl ether (MBTE), mix well, add an appropriate amount of sodium chloride, vortex, centrifuge at 8000 rpm for 10 minutes, and transfer 10 ml of the supernatant into a nitrogen blowing flask. Blow nitrogen down to 1 ml, filter through a 0.22 μm microporous membrane, and collect the filtrate. Detailed preparation and related concentrations are shown in Tables 5 and 6.

[0050] Table 5 Preparation of each solution

[0051] Table 6 Concentrations of each solution

[0052] 1.1.2.8 Test solution Accurately weigh 1.36506 g of the dry suspension and place it in a 50 ml centrifuge tube. Accurately transfer 2 ml of dimethyl sulfoxide and 5 ml of internal standard stock solution 3. Vortex for 30 s. Then accurately transfer 13 ml of ultrapure water and sonicate until no solid particles are visible. Add 10 ml of methyl tert-butyl ether and mix well. Add an appropriate amount of sodium chloride, vortex, and centrifuge at 8000 r / min for 10 min. Take 10 ml of the supernatant and place it in a nitrogen blowing flask. Blow nitrogen down to 1 ml and filter through a 0.22 μm microporous membrane. Collect the filtrate. The second sample weighs 1.36436 g.

[0053] 1.2 Specific Verification Content 1.2.1 System Applicability and Specificity 1.2.1.1 Solution Preparation Blank solution: Prepared in the same way as the blank solution under section 1.1.2.7.

[0054] Reference solution: Prepared in the same way as the reference solution under section 1.1.2.7.

[0055] Limit of quantitation solution: Prepared in the same way as the limit of quantitation solution under section 1.1.2.7.

[0056] 100% spiked test solution: Accurately weigh 1.36346 g of dry suspension and place it in a 50 ml centrifuge tube. Accurately transfer 2 ml of dimethyl sulfoxide, 5 ml of internal standard mixed stock solution 3, and 5 ml of reference standard mixed stock solution 2. Vortex for 30 s. Accurately transfer 13 ml of ultrapure water and sonicate until no solid particles are visible. Add 5 ml of methyl tert-butyl ether and mix well. Add an appropriate amount of sodium chloride. Vortex and centrifuge at 8000 r / min for 10 min. Take 10 ml of the supernatant and place it in a nitrogen blowing flask. Blow nitrogen down to 1 ml and filter through a 0.22 μm microporous membrane. Collect the filtrate.

[0057] 1.2.1.2 Verification Results The blank solution did not detect the three target peaks. In the system suitability solution, after six consecutive injections, the RSD of the ratio of the area of ​​the three target peaks to the area of ​​the internal standard peak was 0.53%–0.71%, all less than 20%. In the re-injection solution, the RSD of the ratio of the area of ​​the three target peaks to the area of ​​the internal standard peak, and the ratio of the corresponding target peak areas measured in the first six reference solutions, were 0.48%–2.70%, all less than 20%. The retention times of the three target peaks in the reference solution and the 100% spiked test solution were essentially the same.

[0058] This method has good system applicability and specificity. The results are shown in Tables 7 to 9.

[0059] Table 7 System Suitability Solution Test Results

[0060] Table 8. System Suitability Solution Backflow Test Results

[0061] Table 9 Specificity Test Results

[0062] 1.2.2 Limit of Quantitation and Limit of Detection 1.2.2.1 Solution Preparation Detection limit solution (DL): Prepared in the same way as DL under section 1.1.2.7.

[0063] Limit of Quantification (QL) Solution: Prepared in the same way as QL under section 1.1.2.7.

[0064] 1.2.2.2 Verification Results Six consecutive injections of the limit of quantitation (LOQ) solution (equivalent to the sample concentrations of NDMA 8.00 ng / g, NDIPA 2.20 ng / g, and NPYR 141.52 ng / g) yielded signal-to-noise ratios (S / N) of 10–47 for all three target peaks, all greater than or equal to 10; the RSDs of the peak areas of all three target peaks ranged from 2.62% to 2.99%, all less than 20%; and the RSDs of the retention times of all three target peaks ranged from 0.01% to 0.04%, all less than 2%. One injection of the limit of detection (LOD) solution (equivalent to the sample concentrations of NDMA 4.00 ng / g, NDIPA 1.10 ng / g, and NPYR 70.78 ng / g) yielded S / Ns of 12–30 for all three target peaks, all greater than 3. Results are presented in Tables 10–12.

[0065] Table 10 Results of quantitative peak area quantification

[0066] Table 11 Results of retention time at the limit of quantitation

[0067] Table 12. Signal-to-noise ratio results for limit of quantitation and limit of detection.

[0068] 1.2.3 Linearity and Range The linearity of the three target peaks in the range of 10% to 200% limit concentration was investigated.

[0069] 1.2.3.1 Solution Preparation Linear solutions L1~L6: Prepared in the same way as L1~L6 under section 1.1.2.7.

[0070] 1.2.3.2 Verification Results Within the concentration limit range of 10% to 200%, the linear correlation coefficient r ranged from 0.9992 to 0.9995, all greater than 0.99; the ratio of the absolute value of the Y-axis intercept to the 100% response value ranged from 1.07% to 3.66%, all within 25%. This method exhibits good linearity and range. The results are shown in Table 13, and the linear spectrum is as follows. Figures 2-4 As shown.

[0071] Table 13 Results of the linearity test

[0072] 1.2.4 Accuracy 1.2.4.1 Solution Preparation Test solution: Prepared as described in section 1.1.2.8. Prepare two parallel solutions. Weigh 1.36506 g and 1.36436 g respectively.

[0073] 10% Spiked Test Solution (ACCL): Accurately weigh 1.36505 g of dry suspension and place it in a 50 ml centrifuge tube. Accurately transfer 2 ml of dimethyl sulfoxide, 5 ml of internal standard mixed stock solution 3, and 0.5 ml of reference standard mixed stock solution 2. Vortex for 30 s. Accurately transfer 13 ml of ultrapure water and sonicate until no solid particles are visible. Add 9.5 ml of methyl tert-butyl ether, mix well, add an appropriate amount of sodium chloride, vortex, and centrifuge at 8000 r / min for 10 min. Take 10 ml of the supernatant and place it in a nitrogen blowing flask. Blow nitrogen down to 1 ml, filter through a 0.22 μm microporous membrane, and collect the filtrate. Prepare three parallel aliquots. The remaining two aliquots weigh 1.36533 g and 1.36244 g, respectively.

[0074] 100% spiked test solution (ACCM): Prepared in the same manner as described in section 1.1. Prepare three parallel portions using the same method. Weigh the remaining two portions to 1.36282 g and 1.36293 g, respectively.

[0075] 150% Spiked Test Solution (ACCH): Accurately weigh 1.36227 g of dry suspension and place it in a 50 ml centrifuge tube. Accurately transfer 2 ml of dimethyl sulfoxide, 5 ml of internal standard mixed stock solution 3, and 7.5 ml of reference standard mixed stock solution 2. Vortex for 30 s. Accurately transfer 13 ml of ultrapure water and sonicate until no solid particles are visible. Add 2.5 ml of methyl tert-butyl ether, mix well, add an appropriate amount of sodium chloride, vortex, and centrifuge at 8000 r / min for 10 min. Take 10 ml of the supernatant and place it in a nitrogen blowing flask. Blow nitrogen down to 1 ml, filter through a 0.22 μm microporous membrane, and collect the filtrate. Prepare three parallel aliquots. The remaining two aliquots weigh 1.36177 g and 1.36278 g, respectively.

[0076] 1.2.4.2 Verification Results At the 10%, 100%, and 150% limit concentration levels, the recoveries of the three target analytes ranged from 92.07% to 118.21%, all within the range of 70% to 130%. The RSDs for each recovery level and the overall recovery at the three concentration levels ranged from 0.78% to 10.25%, all less than 20%. The 95% confidence intervals for the recoveries of the three target analytes were 74.76% to 128.14%, all within the range of 70% to 130%. This method exhibits good accuracy. The test results for the sample are shown in Table 14, and the accuracy results are shown in Tables 15 to 17.

[0077] Table 14 Test Results of the Samples

[0078] Table 15 Accuracy Results for N-Nitrosaminodimethylamine

[0079] Table 16 Accuracy Results for N-Nitrosaminodiisopropylamine

[0080] Table 17 Accuracy Results of N-Nitropyrrolidine

[0081] 1.2.5 Precision 1.2.5.1 Repeatability 1.2.5.1.1 Solution Preparation 100% Spiked Test Solution (ACCM): Prepared in the same manner as the 100% spiked test solution under section 1.2.1.1. Prepare 6 parallel solutions, 3 of which are shared with the 100% spiked test solution under section 1.2.4.1. The sample weights of the remaining 3 solutions are 1.36379 g, 1.36378 g, and 1.36224 g, respectively.

[0082] 1.2.5.1.2 Verification Results Analyst 1 determined that the RSDs of the recoveries of the three target peaks in six 100% concentration-level spiked test solutions ranged from 1.54% to 3.00%, all less than 20%. The method exhibits good repeatability. Results are shown in Table 18.

[0083] 1.2.5.2 Intermediate Precision 1.2.5.2.1 Solution Preparation Analyst 2 prepared blank and reference solutions at different times according to section 1.1.2.7, weighing 20.32 mg of NDMA and 21.58 mg of NPYR; prepared limit solution according to section 1.1.2.7; prepared two parallel test solutions according to section 1.1.2.8, weighing 1.36714 g and 1.36301 g respectively; and prepared six parallel 100% spiked test solutions according to section 1.2.1.1, weighing 1.36869 g, 1.36607 g, 1.36670 g, 1.36699 g, 1.36672 g, and 1.36618 g respectively.

[0084] 1.2.5.2.3 Verification Results Analyst 2 determined the RSDs of the recoveries of the three target peaks in six 100% spiked test solutions to be 1.24%–2.94%, all less than 20%. Combined with repeatability data, the RSDs of the recoveries of the three target peaks in twelve 100% spiked test solutions were calculated to be 1.67%–2.73%, all less than 20%. The upper confidence limits for the RSDs of the three target peak recoveries were 2.60%–4.24%, all less than 20%.

[0085] The method showed good intermediate precision. The results are shown in Table 18.

[0086] Table 18 Results of Intermediate Precision Test

[0087] 1.2.6 Solution stability 1.2.6.1 Solution Preparation Reference solution: Used in conjunction with the reference solution described in section 1.2.1.1.

[0088] 100% spiked test solution: Used in conjunction with the 100% spiked test solution under section 1.2.1.1.

[0089] 1.2.6.2 Determination Method The reference solution and the 100% spiked test solution were injected and analyzed at room temperature at different times. The chromatograms were recorded to examine the stability of the reference solution and the 100% spiked test solution at different time points.

[0090] 1.2.6.3 Verification Results The reference solution and the 100% spiked test solution were incubated at room temperature for 51.5 h and 44.5 h, respectively. The ratios of the concentrations of the three target peaks in the reference solution and the spiked test solution to the concentrations measured at 0 h were 89.88%–106.51% and 89.07%–111.36%, respectively, both within the range of 80%–120%. The reference solution was stable at room temperature for 51.5 h, and the 100% spiked test solution was stable at room temperature for 44.5 h. The results are shown in Tables 19 and 20.

[0091] Table 19 Results of stability study of reference solution

[0092] Table 20 Results of stability study of 100% spiked test solution

[0093] MS quantitative chromatogram as follows Figures 5-9 As shown.

[0094] The analytical method established in this invention can be used to detect NDMA, NDIPA, and NPYR in mapasxavir dry suspension.

[0095] Experimental Example 2: Comparison of Existing Capsule Formulation Methods The same batch of mapasixavir dry suspension was used, and three spiking concentrations were set: low level spiking concentration, medium level spiking concentration, and high level spiking concentration (10%, 100%, and 150%, respectively). The reference standard, nitrogen blowing, volume adjustment, membrane filtration, and instrument detection parameters were kept consistent in both groups of experiments, with only the pretreatment process being different.

[0096] 2.1 Comparative Example: Existing Conventional Pretreatment Methods for Capsules Referring to the standard pretreatment process for capsules in existing technologies, follow these steps: Test solution: Take the dry suspension sample into a centrifuge tube, add 5 ml of water, 1 ml of internal standard stock solution with a concentration of 62.5 ng / ml, and 9 ml of dichloromethane in sequence, mix well, and centrifuge. Transfer 8 ml of the lower organic phase, add methanol, concentrate by nitrogen blowing to about 0.5 ml, and then make up to 1 ml with dichloromethane. Filter through a 0.22 μm microporous membrane to obtain the test solution.

[0097] Spiked test solution: Take the dry suspension sample into a centrifuge tube, add 5 ml of water, 1 ml of internal standard stock solution with a concentration of 62.5 ng / ml, 0.25–3.75 ml of mixed reference stock solution (with NDMA concentration of 9.08 ng / ml, NDIPA concentration of 2.5 ng / ml, and NPYR concentration of 160.7 ng / ml), and 5.25–8.75 ml of dichloromethane sequentially. Mix well and centrifuge. Transfer 8 ml of the lower organic phase, add methanol, and concentrate under nitrogen blowing to approximately 0.5 ml. Then, bring the volume to 1 ml with dichloromethane and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0098] Experimental Phenomena and Results: During the extraction stage, the two phases separated well. However, the matrix of the excipients in the mapasixavir dry suspension was complex, and some excipients entered the dichloromethane layer during extraction. After nitrogen blowing concentration, the system reached a supersaturated state, resulting in a large amount of crystal precipitation. This crystal precipitation not only disrupted the composition of the test system but also easily introduced matrix interference, leading to increased baseline noise. The solution filtered through a 0.22 μm microporous membrane was used to determine the spiked samples at low, medium, and high spiking concentrations. The recovery rate of the target analyte spiked samples ranged from 86.28% to 493.76%, significantly exceeding the allowable quantitative range of 70% to 130%, indicating distorted detection results. Therefore, this method is not suitable for the detection of mapasixavir dry suspension.

[0099] 2.2 Experimental group: The pretreatment method of Experiment Example 1 was used. Take samples of mapasixavir dry suspension from the same batch and prepare 10%, 100%, and 150% spiked sample solutions according to the complete pretreatment steps described in 1.1.2.4, including solvent system, sodium chloride salting-out, high-speed centrifugation at 8000 r / min, nitrogen blowing concentration, and membrane filtration. Inject the samples for testing.

[0100] Experimental Results: The solvent system selected in Example 1 ensured uniform dispersion of the dry suspension powder. Combined with salting-out and high-speed centrifugation, it achieved complete separation of the organic and aqueous phases. No crystallization occurred during the concentration process, and the target analyte remained stably present in the solution system. The recoveries of spiked samples at various concentrations all fell within the acceptable range of 70%–130%, with no matrix interference, good peak shape, and good detection precision and accuracy, enabling stable and precise quantification.

[0101] 2.3 Comparison Conclusion The solvent system of the standard pretreatment process for capsules in the existing technology is not suitable for mapasxavir dry suspension. Crystallization is easily generated during the concentration process, which leads to abnormal recovery rate and serious baseline interference, and cannot meet the detection requirements.

[0102] This invention improves the solubility of the target analyte and completely solves the problem of concentration and crystallization by redesigning the extraction solvent and introducing a synergistic process of sodium chloride salting-out and high-speed centrifugation. It further demonstrates that this invention is a customized detection method specifically for mapasxavir dry suspension, exhibiting outstanding dosage form specificity.

[0103] Experiment Example 3: Comparison of Sodium Chloride Salting-out and High-Speed ​​Centrifugation The same batch of dry suspension samples were taken, and all operations, reagent dosages, and instrument parameters before extraction were completely consistent according to the method in Experiment Example 1. After the extraction of methyl tert-butyl ether, four post-treatment experiments were set up. The system's layering state and emulsification status were used as preliminary judgment indicators, and only solutions with good layering and normal sampling were subjected to subsequent nitrogen blowing, filtration, and sample injection testing.

[0104] Experiment 1: No sodium chloride was added, and no high-altitude centrifugation at 8000 r / min was performed.

[0105] After extraction, the system was left to stand. The separation rate was extremely slow, the interface between the two phases was blurred, and there was obvious emulsification. Sampling conditions were not met, so subsequent nitrogen blowing, filtration, and sample injection were not carried out.

[0106] Experiment 2: Add sodium chloride for salting out, but do not perform high-altitude centrifugation at 8000 r / min.

[0107] Sodium chloride can play a certain role in demulsification, but the system still separates into two phases slowly by natural settling. The organic phase is mixed with the aqueous phase, and the separation of the two phases is incomplete, which does not meet the requirements of the test solution. Therefore, subsequent nitrogen blowing, filtration and sample injection testing were not carried out.

[0108] Experiment 3: No sodium chloride was added, but high-speed centrifugation at 8000 r / min was performed.

[0109] After high-speed centrifugation at 8000 r / min for 10 min, the system layering speed increased, the emulsification phenomenon was alleviated, and the organic phase could be separated. After being successively purged with nitrogen and filtered through a 0.22 μm filter membrane, the solution was prepared as the test solution and injected for detection.

[0110] Test results: Some target objects showed significant damage, and the instrument response values ​​were low (see the table below for specific response value data).

[0111] Experiment 4: Add sodium chloride for salting out, then perform high-altitude centrifugation at 8000 r / min (Experiment Example 1 scheme). After sodium chloride salting-out combined with high-speed centrifugation, the emulsion was completely broken down, and the two phases were rapidly and thoroughly separated, with the organic phase becoming clear and transparent. Subsequent pretreatment was then completed before sample injection and analysis.

[0112] The test results are shown in Table 21. The target substance was fully extracted, and the overall instrument response value was significantly higher than that of Experiment 3.

[0113] Table 21

[0114] Based on the combined experimental phenomena and test results of the four groups, we can conclude that: Relying solely on natural settling (Experiment 1, Experiment 2), regardless of whether sodium chloride is added, it is impossible to achieve effective and rapid separation of the organic phase and the aqueous phase. The system exhibits poor emulsification and stratification, making it impossible to prepare qualified test solutions and thus completely hindering detection. Although basic stratification and sample injection can be achieved by using high-speed centrifugation alone without salting out (Experiment 3), the lack of salting out results in a large amount of target analyte remaining in the aqueous phase, leading to insufficient extraction efficiency and unsatisfactory detection response. In Experiment 1, when sodium chloride salting-out was used in combination with high-speed centrifugation (Experiment 4), the two formed a significant synergistic effect: sodium chloride reduced the solubility of the target substance in the aqueous phase and broke up the emulsion base, while high-speed centrifugation further enhanced the separation effect of the two phases, fundamentally solving the problems of slow layering and incomplete extraction.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting nitrosamine impurities in mapasixavir dry suspension, characterized in that, This includes using gas chromatography-tandem mass spectrometry to detect nitrosamine impurities in reference solutions, test solutions, and blank solutions, using the ratio of the chromatographic peak area of ​​nitrosamine impurities to the chromatographic peak area of ​​their corresponding internal standard as a quantitative parameter, and calculating the content of nitrosamine impurities in the test solution using the internal standard method; The reference solution, test solution, and blank solution all contain the same concentration of internal standard. The test solution was prepared by using mapasixavir dry suspension as the test sample, premixing with dimethyl sulfoxide, adding internal standard and mixing, dissolving in pure water, adding methyl tert-butyl ether and sodium chloride sequentially, vortexing and mixing, and then centrifuging to collect the supernatant and concentrating. The internal standard includes N-nitrosodimethylamine-D6 and / or N-nitrosodi-n-propylamine-D14; The reference standard is a nitrosamine impurity, which includes at least one of N-nitrosodimethylamine, N-nitrosodiisopropylamine, or N-nitrosopyrrolidine.

2. The detection method according to claim 1, characterized in that, In the chromatographic detection, the chromatographic column is a polar capillary column with polyethylene glycol stationary phase; the injection mode is splitless injection, and the injection volume is 2 μl.

3. The detection method according to claim 2, characterized in that, Chromatographic conditions include: The carrier gas is helium (purity ≥99.999%), and the flow rate is 0.95~1.05 mL / min; The injection port temperature is 225~235℃; Column temperature program: Initial temperature 40℃, hold for 1 min, then increase to 120℃ at a rate of 5℃ / min, hold for 2 min, then increase to 240℃ at a rate of 10℃ / min, hold for 4 min.

4. The detection method according to claim 1, characterized in that, Mass spectrometry conditions include: The ion source is an electron bombardment source; The ion source temperature is 250℃; The interface temperature is 250℃; The collision gas is argon. The scanning mode is multi-response monitoring mode.

5. The detection method according to claim 4, characterized in that, The quantitative ion pair for N-nitrosodimethylamine is 74 / 44, and the qualitative ion pair is 74 / 42. The quantitative ion pair for N-nitrosodimethylamine-D6 is 80 / 50, and the qualitative ion pair is 80 / 46. The quantitative ion pair for N-nitrosodiisopropylamine is 130 / 88, and the qualitative ion pair is 130 / 42. The quantitative ion pair of N-nitrosodi-n-propylamine-D14 was 144 / 126, and the qualitative ion pair was 78 / 50. The quantitative ion pair for N-nitrosopyrrolidine is 100 / 70, and the qualitative ion pair is 100 / 55.

6. The detection method according to claim 1, characterized in that, The internal standard method was used for quantification. The peak area ratio of each nitrosamine impurity in the reference solution to that of the internal standard was used as the quantitative parameter. The content of each nitrosamine impurity in the test solution was calculated according to the standard curve method.

7. The detection method according to claim 1, characterized in that, The detection method has a limit of quantification (LOQ) of ≤8.00 ng / g for N-nitrosodimethylamine, a limit of quantification (LOQ) of ≤2.20 ng / g for N-nitrosodiisopropylamine, and a limit of quantification (LOQ) of ≤141.52 ng / g for N-nitrosopyrrolidine. The linear correlation coefficient r is ≥0.99 within the concentration range of 10% to 200%.

8. The detection method according to claim 1, characterized in that, In the test sample solution, for every 1.2258 g to 1.4982 g of test sample, add 2.0 to 4.0 mL of dimethyl sulfoxide, 10.0 to 15.0 mL of pure water, 15.0 to 21.0 mL of methyl tert-butyl ether, and 1.0 to 3.0 g of sodium chloride. The supernatant collection includes collecting 2 / 3 of the total volume of the supernatant. The concentration includes concentrating to 1 / 10 of the volume.

9. The detection method according to claim 8, characterized in that, The vortex duration is 30~60s; Concentration is performed using nitrogen blowing, with a concentration temperature ≤25℃; Centrifugation conditions include centrifugation at 8000 rpm for ≥5 min.

10. The detection method according to claim 9, characterized in that, The reference solution was prepared by diluting nitrosamine impurities with methyl tert-butyl ether and following the same method as the test solution. The reference solution contains solutions with N-nitrosodimethylamine concentrations of 36.32–145.34 ng / ml, N-nitrosodiisopropylamine concentrations of 10–40 ng / ml, and N-nitrosopyrrolidine concentrations of 642.5–2570 ng / ml. The blank solution is prepared without adding the test sample, following the same method as the test sample solution. The concentration of the internal standard in the reference solution, test solution, and blank solution is 25~100 ng / ml.

Images