Method for detecting related substances in (3S, 5S)-3-Boc-amino-5-methylpiperidine

Abstract

The application discloses a detection method of related substances in (3S, 5S)-3-Boc-amino-5-methylpiperidine. The related substances are detected by using a gas chromatography method. The detection method provided by the application solves the interference problem of other impurities possibly introduced in the process. The application researches and screens gas phase lining pipes of different materials, capillary columns of different stationary liquids, different sample injection procedures, sample injection bottle materials and sample dispensing droppers, provides an optimized detection method, and proposes solutions to the problems of poor reproducibility, adsorption and leaching in the detection process, so that the accuracy of the detection result is ensured.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis and detection technology, specifically relating to a method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Nemonoxacin malate, chemically named 7-[(3S,5S)-3-amino-5-methylpiperidin-1-yl]-1-cyclopropyl-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid malate hemihydrate, is the world's first fluoroquinolone antibacterial drug developed by Procter & Gamble. In its synthesis, (3S,5S)-3-Boc-amino-5-methylpiperidine is an essential starting material (CN101045725B; Synthesis of Nemonoxacin Malate, Chen Gang et al.). The structural formula of (3S,5S)-3-Boc-amino-5-methylpiperidine is as follows: .

[0004] (3S,5S)-3-Boc-amino-5-methylpiperidine contains two chiral carbons, inevitably resulting in the coexistence of isomer impurities. These isomer impurities and other related substances directly affect subsequent reactions in the synthesis of nemonoxacin malate, impacting product purity. Therefore, it is necessary to strictly control the content of related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine.

[0005] However, no publicly available patents or other literature reports methods for the detection and control of its impurities. Furthermore, this compound is a piperidine derivative; while piperidine compounds have simple structures, they are highly reactive, exhibiting strong adsorption and leaching abilities towards organic matter, making sample preparation difficult during analytical method development. These issues make the detection of related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine a challenge.

[0006] The liner is a key component of gas chromatography. After the sample enters the instrument through the syringe, the components in the sample first come into contact with the liner and the glass wool inside the liner during the vaporization process. Liner types include glass wool split / splitless liners, short, highly inert splitless liners with glass wool, and glass frit split liners. Li Siyuan, in his paper "Determination of Seven Dimethylcyclosiloxanes in Cosmetics by Gas Chromatography-Tandem Mass Spectrometry," points out that the liner affects residues and can cause peak tailing. No research reports have been published on the influence of the liner on the detection of piperidine compounds. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine, wherein the related substances are detected by gas chromatography, and the chromatographic conditions of the gas chromatography are as follows: A capillary column with 100% dimethylpolysiloxane (or similar polarity) as the stationary phase was used as the chromatographic column; the column flow rate was 1.8~2.2 ml per minute; The split ratio is 15:1 to 25:1; The heating program is as follows: the initial temperature is 45-60℃, maintained for 2-3 minutes, then the temperature is increased to 150-170℃ at a rate of 7-9℃ per minute, maintained for 7-9 minutes, and then the temperature is increased to 290-300℃ at a rate of 14-16℃ per minute, maintained for 8-12 minutes. The injection port temperature is 255~265℃; the detector is a flame ionization detector (FID) with a detector temperature of 315~325℃.

[0009] In some embodiments of the present invention, the sample injection liner is a glass frit liner.

[0010] Comparative analysis revealed that the repeatability of test results using glass frit liner was significantly better than that of other types of liner.

[0011] In some embodiments of the present invention, the chromatographic column is an Agilent DB-1, 0.32 mm × 30 m, 0.5 μm. Compared with other chromatographic columns, this column provides better resolution for detecting various impurities.

[0012] In some embodiments of the present invention, the preparation of the sample is also included, wherein the test solution is prepared with acetonitrile, the concentration of the test solution is 20±2 mg / ml, and the concentration of the control solution is 40±4 μg / ml.

[0013] In some embodiments of the present invention, the sample is stored in a glass container for no more than 5 minutes. Further, the container includes a sample infusion bottle.

[0014] Furthermore, the sample is specifically a self-control solution. The self-control solution is injected immediately after preparation.

[0015] The sample vial is made of high borosilicate glass.

[0016] Plastic droppers should not be used during sample preparation to avoid introducing leachate from the plastic into the test results.

[0017] In some embodiments of the present invention, a glass dropper is used in the sample preparation process.

[0018] In some embodiments of the present invention, the initial temperature is 50°C, maintained for 3 minutes, then increased to 160°C at a rate of 8°C per minute, maintained for 8 minutes, and then increased to 300°C at a rate of 15°C per minute, maintained for 10 minutes. Under these conditions, the separation of impurities ii-9 / ii-10 from the main peak is better compared to other conditions.

[0019] In some embodiments of the present invention, the injection port temperature is 260°C.

[0020] In some embodiments of the present invention, the detector is a flame ionization detector (FID) with a detector temperature of 320°C.

[0021] The following technical problems were overcome during the research and development of this technical solution: 1. This invention provides a method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine, which solves the problems of easy interference and poor accuracy of the detection of piperidine impurities by high performance liquid chromatography.

[0022] 2. As a piperidine compound, improper sample preparation and container use can cause fluctuations and changes in the chromatographic peak area, affecting the reproducibility and accuracy of the detection results. To address this issue, this invention researched and screened gas chromatography liners of different materials, capillary columns with different stationary phases, different temperature programs, sample vial materials, and sample droppers, providing an optimized detection method. Furthermore, solutions were proposed to address issues such as poor reproducibility, adsorption, and leaching during the detection process, ensuring the accuracy of the detection results.

[0023] 3. In this method, the separation between the peaks of each component is good. The method was validated under these conditions, and it showed high accuracy, precision, and sensitivity, as well as good solution stability and specificity, providing a reliable detection method for related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1 This is the chromatogram of batch Y24034301 of the test sample in Example 1.

[0026] Figure 2This is a comparison chart of the effects of using different droppers in Example 2; the charts from top to bottom are: solvent using a plastic dropper, spiked sample using a plastic dropper, solvent using a glass dropper, and spiked sample using a glass dropper.

[0027] Figure 3 This is the specificity test chromatogram in Example 3; wherein, from bottom to top, the chromatogram consists of solvent (acetonitrile), reference solution ①, reference solution ②, reference solution ③, spiked test solution ①, spiked test solution ②, spiked test solution ③, and test solution.

[0028] Figure 4 The chromatogram of the test solution was obtained by using DB-35 in Comparative Example 1.

[0029] Figure 5 This is a chromatogram of the spiked test solution in Comparative Example 2 with different gradient elution programs. Detailed Implementation

[0030] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] The present invention will be further described below with reference to the embodiments.

[0032] The synthetic route for (3S,5S)-3-Boc-amino-5-methylpiperidine is as follows:

[0033] Starting with ii-1, pyridine (ii-3) was used as an acid-binding agent and a substitution reaction was carried out in the presence of cyanuric chloride to generate intermediate ii-2. Benzylamine (BnNH2) was added to cyclize and demethylate the intermediate to generate (3S,5S)-3-Boc-amino-5-methylpiperidine. The isomers were separated and purified using the chiral resolving agent di-p-methylbenzoyl tartaric acid (DTTA). Then, sodium hydroxide was added to remove tartaric acid, yielding crude (3S,5S)-3-Boc-amino-5-methylpiperidine, which was then purified to obtain the refined product.

[0034] (3S,5S)-3-Boc-amino-5-methylpiperidine contains two chiral carbons. Based on the analysis of its structure and synthetic route, the possible impurities in (3S,5S)-3-Boc-amino-5-methylpiperidine are shown in the table below:

[0035] The analysis of each impurity is as follows: (1) Starting materials: Impurity ii-1: The starting material for the production of (3S,5S)-3-Boc-amino-5-methylpiperidine may remain in (3S,5S)-3-Boc-amino-5-methylpiperidine due to incomplete reaction, and needs to be investigated.

[0036] (2) Intermediate: Impurity ii-2: An intermediate remaining from incomplete reaction during the preparation of (3S,5S)-3-Boc-amino-5-methylpiperidine, which may remain in (3S,5S)-3-Boc-amino-5-methylpiperidine and requires further investigation.

[0037] (3) Process impurities: Impurity ii-3: As an acid-binding agent used in the preparation of (3S,5S)-3-Boc-amino-5-methylpiperidine, it may remain in (3S,5S)-3-Boc-amino-5-methylpiperidine and needs to be investigated.

[0038] Impurity ii-4: generated by the reaction of intermediate ii-2 with the cyclizing reagent benzylamine.

[0039] Impurity ii-5: Benzylamine, the cyclizing agent used in the preparation of (3S,5S)-3-Boc-amino-5-methylpiperidine, may remain in (3S,5S)-3-Boc-amino-5-methylpiperidine and needs to be investigated.

[0040] Impurity ii-8: Impurities generated during the removal of methyl groups in the preparation of (3S,5S)-3-Boc-amino-5-methylpiperidine may remain in (3S,5S)-3-Boc-amino-5-methylpiperidine and require further investigation.

[0041] .

[0042] Impurities ii-9 and ii-10 are diastereomers generated during the preparation of (3S,5S)-3-Boc-amino-5-methylpiperidine, which may remain in (3S,5S)-3-Boc-amino-5-methylpiperidine and require further investigation.

[0043] Example 1 1. Solution preparation Test solution: Take about 200 mg of this product, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0044] Control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with acetonitrile to prepare a solution containing approximately 40 μg per ml.

[0045] Sensitivity solution: Accurately measure 5 ml of the control solution and place it in a 20 ml volumetric flask. Dilute to the mark with acetonitrile and shake well.

[0046] In this embodiment, the liner is made of glass frit (Agilent 5190-5105), the volumetric bottle is made of Tianbo, and the injection bottle is made of Hamai high borosilicate glass.

[0047] 2. Chromatographic conditions and system suitability The chromatographic column used was a capillary column with 100% dimethylpolysiloxane (or a similar polarity) as the stationary phase (Agilent DB-1, 0.32 mm × 30 m, 0.5 μm or equivalent column is recommended); the initial temperature was 50 °C, held for 3 minutes, then increased to 160 °C at a rate of 8 °C per minute, held for 8 minutes, and then increased to 300 °C at a rate of 15 °C per minute, held for 10 minutes; the injection port temperature was 260 °C; the detector was a flame ionization detector (FID) with a detector temperature of 320 °C; the column flow rate was 2.0 mL per minute; a glass frit liner was used with a split ratio of 20:1; and the injection volume was 1 μL.

[0048] For system suitability requirements, the theoretical plate number in the reference solution chromatogram, calculated based on the (3S,5S)-3-Boc-amino-5-methylpiperidine peak, should be no less than 5000. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be greater than 10.

[0049] 3. Determination method Accurately measure the test solution and the control solution, place them in high borosilicate glass vials (brands: Hammer, Agilent, or Waters), inject them into the gas chromatograph within 5 minutes, record the chromatograms, calculate the impurity content and the total impurity using the principal component self-comparison method with correction factors.

[0050] Calculation formula:

[0051] In the formula A 样1 The peak areas of known impurities in the chromatogram of the test sample solution; A 对 The peak area of ​​the main peak in the chromatogram of the control solution; f The correction factor is 1 for known impurities and 1 for unknown impurities.

[0052]

[0053] The above methods were used to detect related substances in batches of crude product Y24034301, refined products 2Y24090904, and 2Y24092102, respectively. The results are as follows: Table 1. Results of Related Substances Detection in Samples

[0054] Note: "ND" means less than the detection limit.

[0055] Example 2 1. Comparison of the performance of different linings The self-control solution was detected using the chromatographic conditions of Example 1, with comparisons made using glass frit lined tubes and glass wool lined tubes. The experimental results are shown in the table below.

[0056] Table 2. Comparison results using glass frit cores and glass liner tubes.

[0057] Table 2 shows that the repeatability of the glass frit liner is significantly better than that of the glass wool liner. This is presumably because the inner tubes in the glass frit act as a barrier, protecting the analytical column from non-volatile contaminants and increasing the volatilization surface area. The uniform pore structure ensures that the liner maintains consistent compound volatilization while trapping non-volatile contaminants.

[0058] 2. Comparison of the effects of different injection processes 1) During the experiment, the peak areas of the same self-control solution were compared after being prepared in volumetric flasks and then transferred to injection vials of different brands and left for different periods of time before injection. The results are summarized in Table 3: Table 3 Comparison of results from different injection processes

[0059] Volumetric flask material: high borosilicate glass.

[0060] Sample vial materials: Hamer, high borosilicate glass; Agilent, Type 1 borosilicate glass (part number: 5190-9591, conforming to ISO 9001 standard); Waters, Type 1 borosilicate glass.

[0061] The comparison results show that if the sample vial is left for a long time, the peak area gradually decreases. Studies have found that volumetric flasks and sample vials from different manufacturers contain metal ions. Furthermore, since this product is a piperidine-based substance, the presence of nitrogen in the piperidine structure results in high activity. If the storage time is too long, the metal residue in the sample vial will adsorb (3S,5S)-3-Boc-amino-5-methylpiperidine, which will affect the results.

[0062] Based on this finding, it is stipulated that the self-control solution can only be transferred from the volumetric flask to the injection bottle (high borosilicate) within 5 minutes before injection to ensure the accuracy of the results.

[0063] 2) To verify the above phenomena, prepare a solution with the same concentration as the control solution in Example 1. The specific method is as follows: Accurately measure 1 ml of the test solution and place it in a 20 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 1 ml of this solution and place it in a 25 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. Measure 1 ml of each solution into a sample vial (Agilent). Add 0.1 ml of 3 mmol / L copper chloride to one vial, and do not add any to the other.

[0064] The chromatographic conditions of Example 1 were used for testing, and the results of the main peak area test are shown in Table 4. Table 4 Comparison of detection results for copper chloride solution

[0065] The above experiments show that the peak area of ​​the main peak decreases significantly with prolonged storage time after the addition of metal ions. The likely reason is that the nitrogen-containing piperidine structure forms a chelate with the metal ions after prolonged storage, affecting the normal test results.

[0066] 3. Comparison of the effects of different droppers The test solution was analyzed using the chromatographic conditions provided in Example 1. A comparison was made between using plastic droppers and glass droppers. The experimental results are shown below. Figure 2 .

[0067] Depend on Figure 2 It is evident that if a plastic dropper is used during sample preparation, piperidine will leach organic matter from the dropper. According to literature review, plastics are mainly polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), and polycarbonate (PC), etc. Plastic droppers are primarily made of PE, which may contain residual contaminants such as ethylene, polyethylene terephthalate, fabric softener oleamide, mold release agents, and long-chain alkanes, which will elute in the gas phase. Therefore, samples should not come into contact with plastic products to avoid interference from leachate on the test results.

[0068] In summary, when testing piperidine compounds, attention should be paid to the selection of containers that come into contact with the sample. This invention has determined the method by selecting different materials for gas phase liners, capillary columns with different stationary liquids, different temperature programs, sample vial materials, and sample droppers, thus ensuring the accurate inspection of samples and impurities therein.

[0069] Example 3 The following methodological validation experiments were conducted using the chromatographic conditions described in Example 1: 1. System suitability and specificity test Acetonitrile was added to different volumetric flasks to dissolve and dilute the impurities to prepare stock solutions containing 0.4 mg / ml of impurity ii-1, impurity ii-2, impurity ii-3, impurity ii-4, impurity ii-5, and impurity ii-8.

[0070] Acetonitrile was added to different volumetric flasks to dissolve and dilute the impurity ii-9 reference standard stock solution and the impurity ii-10 reference standard stock solution containing 0.3 mg / ml.

[0071] For each impurity positioning solution: Take 0.5 ml of each of the following reference solutions: impurity ii-1, impurity ii-2, impurity ii-3, impurity ii-4, impurity ii-5, impurity ii-8, impurity ii-9, and impurity ii-10. Place them in eight different 5 ml volumetric flasks, dilute to the mark with acetonitrile, and shake well.

[0072] Reference solution ①: Accurately measure 0.5 ml each of impurity ii-2 reference stock solution, impurity ii-3 reference stock solution, impurity ii-4 reference stock solution and impurity ii-9 reference stock solution, place them in the same 5 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0073] Reference solution ②: Accurately measure 0.5 ml each of impurity ii-5 reference stock solution, impurity ii-8 reference stock solution and impurity ii-10 reference stock solution, place them in the same 5 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0074] Reference solution ③: Accurately measure 0.5 ml of impurity ii-1 reference stock solution, place it in a 5 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0075] Test solution: Take about 200 mg of this product, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0076] Spiked test solution ①: Take about 100 mg of this product and place it in a 5 ml volumetric flask. Accurately add 0.5 ml each of the impurity ii-2 reference stock solution, impurity ii-3 reference stock solution, impurity ii-4 reference stock solution, and impurity ii-9 reference stock solution. Dissolve and dilute to the mark with acetonitrile and shake well.

[0077] Spiked test solution ②: Take about 100 mg of this product and place it in a 5 ml volumetric flask. Accurately add 0.5 ml each of impurity ii-5 reference stock solution, impurity ii-8 reference stock solution and impurity ii-10 reference stock solution. Dissolve and dilute to the mark with acetonitrile and shake well.

[0078] Spiked test solution ③: Take about 100 mg of this product and place it in a 5 ml volumetric flask. Accurately add 0.5 ml of impurity ii-1 reference stock solution, dissolve and dilute to the mark with acetonitrile, and shake well.

[0079] Control solution: Accurately measure 1 ml of the spiked test solution ① and place it in a 20 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 1 ml of the solution and place it in a 25 ml volumetric flask. Dilute to the mark with acetonitrile and shake well.

[0080] Sensitivity solution: Accurately measure 5 ml of the control solution and place it in a 20 ml volumetric flask. Dilute to the mark with acetonitrile and shake well.

[0081] Accurately measure the solvent (acetonitrile), sensitivity solution, and control solution, and inject them into the gas chromatograph. The control solution is injected five times consecutively, and the chromatograms are recorded.

[0082] Verification results: After five consecutive injections of the control solution, the RSD of the retention time of the main component peak was 0.006%, less than 1%, and the RSD of the peak area was 7.6%, less than 10%. In the chromatogram of the first control solution, the theoretical plate number of the main component peak was 427,056, greater than 5,000, and the tailing factor was 1.0, less than 1.5. In the chromatogram of the sensitivity solution, the signal-to-noise ratio of the main component peak height was 96, greater than 10, indicating that the method has good system suitability.

[0083] Accurately measure the amounts of each impurity localization solution, reference solution, test solution, and spiked test solution, and inject them separately into the gas chromatograph, recording the chromatograms. The experimental results are shown in Table 5 and... Figure 3 .

[0084] Table 5 Results of specificity test

[0085] Note: " / " does not need to be calculated, and the same applies below.

[0086] The results above show that the solvent (acetonitrile) does not interfere with the detection of impurities ii-1, ii-2, ii-3, ii-4, ii-5, ii-8, ii-9, ii-10, and other individual impurities studied by this method, and baseline separation should be achieved between all component peaks. In the spiked test solution, the theoretical plate number calculated based on the principal component peak is greater than 5000, and the tailing factor of the principal component peak is less than 1.5, indicating that the method has good specificity.

[0087] 2. Linearity and Range Tests Impurity ii-1 reference standard stock solution: Weigh approximately 4 mg of impurity ii-1 reference standard accurately, place it in a 50 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0088] For impurity ii-2 reference standard stock solution, impurity ii-3 reference standard stock solution, impurity ii-4 reference standard stock solution, impurity ii-5 reference standard stock solution, impurity ii-8 reference standard stock solution, impurity ii-9 reference standard stock solution and impurity ii-10 reference standard stock solution: prepare the corresponding impurity reference standard stock solution according to the "System Suitability and Specificity Test" section.

[0089] (3S,5S)-3-Boc-amino-5-methylpiperidine reference standard stock solution ①: Weigh approximately 4 mg of (3S,5S)-3-Boc-amino-5-methylpiperidine reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well.

[0090] (3S,5S)-3-Boc-amino-5-methylpiperidine reference standard stock solution ②: Weigh approximately 4 mg of (3S,5S)-3-Boc-amino-5-methylpiperidine reference standard accurately, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well.

[0091] Note: Due to the high price of impurities ii-1 and ii-2, and in order to save on reference standards, it is not possible to prepare high-concentration impurity reference standard stock solutions. Linear stock solutions are prepared separately to reduce dilution steps.

[0092] Linear stock solution ①: Accurately measure 2 ml each of impurity ii-5 reference stock solution, impurity ii-8 reference stock solution, impurity ii-10 reference stock solution and (3S,5S)-3-Boc-amino-5-methylpiperidine reference stock solution ①, place them in the same 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0093] Linear stock solution ②: Accurately measure 2 ml each of impurity ii-2 reference stock solution, impurity ii-3 reference stock solution and impurity ii-4 reference stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0094] Linear stock solution ③: Accurately measure 2 ml each of impurity ii-9 reference stock solution and (3S,5S)-3-Boc-amino-5-methylpiperidine reference stock solution ①, place them in the same 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0095] The first group of linear solutions 1-2~5 (20%, 60%, 100%, 140%): Accurately measure 0.5 ml, 1.5 ml, 2.5 ml, and 3.5 ml of linear stock solution ① and place them in four different 5 ml volumetric flasks. Dilute to the mark with acetonitrile and shake well.

[0096] Linear solutions 1-6 (200%): Accurately measure linear stock solution ① and inject directly.

[0097] The second group of linear solutions 2-2~5 (20%, 60%, 100%, 140%): Accurately measure 0.5 ml, 1.5 ml, 2.5 ml, and 3.5 ml of linear stock solution ②, place them in four different 5 ml volumetric flasks, dilute to the mark with acetonitrile, and shake well.

[0098] Group 2 linear solutions 2-6 (200%): Accurately measure linear stock solution ② and inject directly.

[0099] The third group of linear solutions 3-2~5 (20%, 60%, 100%, 140%): Accurately measure 0.5 ml, 1.5 ml, 2.5 ml, and 3.5 ml of linear stock solution ③, place them in four different 5 ml volumetric flasks, dilute to the mark with acetonitrile, and shake well.

[0100] Group 3 linear solutions 3-6 (200%): Accurately measure linear stock solution ③ and inject directly.

[0101] The fourth group of linear solutions 4-2~5 (20%, 60%, 100%, 140%): Accurately measure 0.5 ml, 1.5 ml, 2.5 ml, and 3.5 ml of impurity ii-1 reference stock solution and (3S,5S)-3-Boc-amino-5-methylpiperidine reference stock solution ②, place them in four different 5 ml volumetric flasks, dilute to the mark with acetonitrile, and shake well.

[0102] Group 4 linear solutions 4-6 (200%): Accurately measure impurity ii-1 reference stock solution and (3S,5S)-3-Boc-amino-5-methylpiperidine reference stock solution ②, and inject directly.

[0103] Linear solutions 2-6 were used as 20%, 60%, 100%, 140%, and 200% linear solutions of impurity limit concentrations, respectively (as points 2-6 of the linearity test). Appropriate amounts of stock solutions of each impurity reference standard and stock solution of (3S,5S)-3-Boc-amino-5-methylpiperidine reference standard were accurately measured and quantitatively diluted with acetonitrile to prepare solutions with a signal-to-noise ratio of peak height of each component not less than 10, which were used as limit of quantitation solutions. The limit of quantitation solution was used as the first point of linearity.

[0104] Accurately measure the above linear solutions and inject them into the gas chromatograph, recording the chromatograms. Perform linear regression with the concentration C (μg / ml) of each component as the x-axis and the peak area A of each component as the y-axis. Calculate the correction factor for each impurity using the ratio of the slope of the linear relationship curve of (3S,5S)-3-Boc-amino-5-methylpiperidine to the slope of the linear relationship curve of each impurity. The calculation results of the correction factors are shown in Table 6. Table 6 Calculation results of correction factors

[0105] Verification conclusion: The linear equations for each component peak (concentration range, linear correlation coefficient r, and percentage of the absolute value of the intercept equivalent to 100% of the response value) are as follows: (3S,5S)-3-Boc-amino-5-methylpiperidine: A = 0.5753C - 0.5544 (4.0169µg / ml ~ 80.3383µg / ml, 0.9997, 2.5); Impurity ii-5: A = 0.5521C - 2.4743 (3.9402µg / ml ~ 78.8045µg / ml, 0.9955, 1 2.6); Impurity ii-8: A = 0.5568C - 1.3032 (3.9975µg / ml ~ 79.9508µg / ml, 0.9997, 6.2); Impurity ii-10: A = 0.5929C - 0.6883 (2.9677µg / ml ~ 59.3547µg / ml, 0.9996, 4.0); Impurity ii-3: A =0.9579C-3.0832 (4.0459µg / ml~80.9178µg / ml, 0.9996, 8.8); Impurity ii-2: A = 0.7214C-1.4647 (3.7196µg / ml~74.3912µg / ml, 0.9994, 5.3); Impurity ii-4: A =0.9003C+0.0599 (1.9350µg / ml~96.7480µg / ml, 0.9992, 0.2); Impurity ii-9: A =0.7210C-1.1339 (1.6878µg / ml~56.2597µg / ml, 0.9997, 5.5); Impurity ii-1: A = 0.3451C - 1.4519 (4.2188µg / ml ~ 84.3752µg / ml, 0.9993, 11.8).

[0106] The correlation coefficients r of the linear relationships of each component are all greater than 0.99, and the percentage of the absolute value of the intercept corresponding to 100% of the response value is all less than 25%, indicating that the method has good linearity.

[0107] The correction factors for impurities ii-2, ii-5, ii-8, ii-9, and ii-10 are all in the range of 0.8 to 1.2, and they can all be quantitatively calculated using the principal component self-comparison method without correction factors. The correction factors for impurities ii-3 and ii-4 are both less than 0.8, and quantitative calculation using the principal component self-comparison method without correction factors is equivalent to strict control.

[0108] 3. Limit of Quantitation and Limit of Detection Test The limit of quantitation (LOQ) was defined as a signal-to-noise ratio (S / N) ≥ 10 for each component peak, and the limit of detection (LOD) was defined as a S / N ≥ 3. The lowest quantitation concentration (μg / ml) and lowest detection concentration (μg / ml), along with their percentages relative to the test sample concentration and the limit concentration, were reported. Six aliquots of the LOQ concentration solution were prepared and injected sequentially, and the chromatograms were recorded to assess the repeatability of the LOQ test.

[0109] The results of the limit of quantitation and limit of detection tests are shown in Table 7.

[0110] Table 7 Results of tests for limit of quantitation and limit of detection

[0111] The results above show that the limits of quantitation (LOQs) for (3S,5S)-3-Boc-amino-5-methylpiperidine, impurities ii-3, ii-5, ii-8, ii-2, ii-1, and ii-4 are all no greater than 20% of the limit concentration and less than 0.04% of the sample concentration. The limits of quantitation for impurities ii-9 and ii-10 are also no greater than 20% of the limit concentration and less than 0.03% of the sample concentration. The signal-to-noise ratios of the peak heights are all higher than 10.

[0112] Limits of detection: The minimum detectable concentrations of (3S,5S)-3-Boc-amino-5-methylpiperidine, impurities ii-3, ii-5, ii-8, ii-2, ii-1, and ii-4 are all less than 10% of the limit concentration and less than 0.02% of the sample concentration. The minimum detectable concentrations of impurities ii-9 and ii-10 are all less than 10% of the limit concentration and less than 0.015% of the sample concentration. The signal-to-noise ratio of the peak heights is all greater than 3.

[0113] 4. Solution stability test For impurity ii-1 reference stock solution, impurity ii-2 reference stock solution, impurity ii-3 reference stock solution, impurity ii-4 reference stock solution, impurity ii-5 reference stock solution, impurity ii-8 reference stock solution, impurity ii-9 reference stock solution and impurity ii-10 reference stock solution: prepare the corresponding impurity reference stock solution according to the "System Suitability and Specificity Test" section.

[0114] Test solution: Take about 200 mg of this product, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0115] Spiked test solution ①: Take about 100 mg of this product and place it in a 5 ml volumetric flask. Accurately add 0.5 ml each of the impurity ii-2 reference stock solution, impurity ii-3 reference stock solution, impurity ii-4 reference stock solution and impurity ii-9 reference stock solution. Dissolve and dilute to the mark with acetonitrile and shake well.

[0116] Spiked test solution ②: Take about 100 mg of this product and place it in a 5 ml volumetric flask. Accurately add 0.5 ml each of impurity ii-5 reference stock solution, impurity ii-8 reference stock solution and impurity ii-10 reference stock solution. Dissolve and dilute to the mark with acetonitrile and shake well.

[0117] Spiked test solution ③: Take about 100 mg of this product and place it in a 5 ml volumetric flask. Accurately add 0.5 ml of impurity ii-1 reference stock solution, dissolve and dilute to the mark with acetonitrile, and shake well.

[0118] Control solution: Accurately measure 1 ml of the test solution and place it in a 20 ml volumetric flask. Dilute to the mark with acetonitrile and shake well. Accurately measure 1 ml of the test solution and place it in a 25 ml volumetric flask. Dilute to the mark with acetonitrile and shake well.

[0119] Reference solution ①: Accurately measure 0.5 ml each of impurity ii-2 reference stock solution, impurity ii-3 reference stock solution, impurity ii-4 reference stock solution and impurity ii-9 reference stock solution, place them in the same 5 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0120] Reference solution ②: Accurately measure 0.5 ml each of impurity ii-5 reference stock solution, impurity ii-8 reference stock solution and impurity ii-10 reference stock solution, place them in the same 5 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0121] Reference solution ③: Accurately measure 0.5 ml of impurity ii-1 reference stock solution, place it in a 5 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0122] Reference solution ①, reference solution ②, spiked test solution ①, spiked test solution ②, test solution, and reference solution were placed at room temperature and analyzed at 0h, 15h, 39h, and 48h after preparation. Reference solution ③ and spiked test solution ③ were placed at room temperature and analyzed at 0h, 8h, and 24h after preparation. Chromatograms were recorded, and the percentage of peak area of ​​each component in the chromatograms of the reference solution and the reference solution at each time point to the peak area at 0h was calculated. The absolute value of the difference between the content of each impurity in the spiked test solution and the test solution at each time point and the content at 0h was calculated using the peak area normalization method. The experimental results are shown in Tables 8 to 13.

[0123] Table 8. Results of stability tests on reference solution and control solution

[0124] Table 9. Results of stability test on reference solution ③

[0125] Table 10 Results of stability test on spiked test solution ①

[0126] Table 11 Results of stability test on spiked test solution ②

[0127] Table 12 Results of the stability test of the spiked test solution ③

[0128] Table 13 Results of the stability test of the test sample solution

[0129] Verification conclusion: After 48 hours at room temperature, the peak area ratio of each component at each time point to the peak area at 0 h was within the range of 90% to 108%, indicating that the stability of reference solutions ① and ② was good for at least 48 hours at room temperature, the stability of the control solution was good for at least 39 hours at room temperature, and the stability of reference solution ③ was good for at least 24 hours at room temperature.

[0130] The test solutions, spiked test solutions ① and ② were placed at room temperature for 48 hours, and spiked test solution ③ was placed at room temperature for 24 hours. The absolute value of the difference between the content of each known impurity at each time point and the content at 0 h was not greater than 0.02%, the absolute value of the difference between the content of other individual impurities and the content at 0 h was not greater than 0.02%, and the absolute value of the difference between the total amount of impurities and the content at 0 h was not greater than 0.05%. No new impurities exceeding the reporting limit were generated. This indicates that the test solutions, spiked test solutions ① and ② have good stability at room temperature for at least 48 hours, and spiked test solution ③ has good stability at room temperature for at least 24 hours.

[0131] 5. Repeatability test Prepare the reference solutions, spiked test solutions, and control solutions according to the method described in the "System Suitability and Specificity Test" section. Analyst 1 accurately measures the solvent, reference solutions, spiked test solutions, and control solutions, and injects them separately into the gas chromatograph, recording the chromatograms. Calculate the content of each known impurity in the spiked test solution using both the external standard method and the principal component self-comparison method with correction factors. Calculate the content of other individual impurities using the self-comparison method. The results are shown in Tables 14-17.

[0132] Table 14 Results of Repeatability Tests (External Standard Method)

[0133] Table 15 Results of Repeatability Tests (Principal Component Self-Control Method with Correction Factors) - Group 1

[0134] Table 16 Results of Repeatability Tests (Principal Component Self-Control Method with Correction Factors) - Group 2

[0135] Table 17 shows the experimental results comparing the two calculation methods.

[0136] Verification conclusion: In the six spiked test solutions, the RSD of each known impurity was less than 3%, the RSD of other individual impurities was less than 5%, and the RSD of the total impurities was less than 2%. The results of the principal component self-comparison method with correction factor and the external standard method for impurities were basically consistent (the relative difference was not greater than 10%), so the principal component self-comparison method with correction factor can be used for impurity quantification.

[0137] 6. Intermediate precision test Using the same batch of samples, six spiked test solutions and control solutions were prepared by different analysts at different times, following repeatability testing methods. The amounts of each impurity were determined using different gas chromatographs according to repeatability testing methods. The amounts of known impurities were calculated using the principal component self-comparison method with correction factors, and the amounts of other individual impurities were calculated using the self-comparison method. Combining the repeatability test results, a total of 12 results were obtained. The RSD of the detection results for each impurity was calculated to assess the precision of the method.

[0138] Verification conclusions: In the 6 spiked test solutions, the RSD of each known impurity was no greater than 3%, the RSD of other individual impurities was less than 5%, and the RSD of the total impurities was less than 2%. Combining the 6 test results with the 6 results from the repeatability test, for a total of 12 spiked test solutions, the RSD of each known impurity was no greater than 6%, the RSD of other individual impurities was less than 10%, and the RSD of the total impurities was less than 4%, indicating that the method has good intermediate precision.

[0139] 7. Accuracy Test Impurity ii-1 reference standard stock solution: Weigh approximately 4 mg of impurity ii-1 reference standard accurately, place it in a 50 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0140] For impurity ii-2 reference standard stock solution, impurity ii-3 reference standard stock solution, impurity ii-4 reference standard stock solution, impurity ii-5 reference standard stock solution, impurity ii-8 reference standard stock solution, impurity ii-9 reference standard stock solution and impurity ii-10 reference standard stock solution: prepare the corresponding impurity reference standard stock solution according to the "System Suitability and Specificity Test" section.

[0141] Accuracy stock solution ①: Accurately measure 5 ml each of impurity ii-2 reference stock solution, impurity ii-3 reference stock solution, impurity ii-4 reference stock solution, and impurity ii-9 reference stock solution, place them in the same 25 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0142] Accuracy stock solution ②: Accurately measure 5 ml each of impurity ii-5 reference stock solution, impurity ii-8 reference stock solution and impurity ii-10 reference stock solution, place them in the same 25 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0143] Reference solution ①: Accurately measure 1 ml of accuracy stock solution ①, place it in a 2 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0144] Reference solution ②: Accurately measure 1 ml of accuracy stock solution ②, place it in a 2 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0145] Reference solution ③: Accurately measure 2.5 ml of impurity ii-1 reference stock solution, place it in a 5 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0146] Accuracy of the first group of samples 1-1~3 (60%, 100%, 140%): Weigh approximately 100 mg of this product accurately and place it in three different 5 ml volumetric flasks. Accurately add 1.5 ml, 2.5 ml, and 3.5 ml of accuracy stock solution ①, respectively. Dissolve and dilute to the mark with acetonitrile and shake well. Prepare three replicates for each concentration.

[0147] Accuracy of the second group of samples 2-1~3 (60%, 100%, 140%): Weigh approximately 100 mg of this product accurately and place it in three different 5 ml volumetric flasks. Accurately add 1.5 ml, 2.5 ml, and 3.5 ml of accuracy stock solution ②, respectively. Dissolve and dilute to the mark with acetonitrile and shake well. Prepare three replicates for each concentration.

[0148] Accuracy of the third group of samples 3-1~3 (60%, 100%, 140%): Weigh approximately 100 mg of this product accurately and place it in three different 5 ml volumetric flasks. Accurately add 1.5 ml, 2.5 ml, and 3.5 ml of impurity ii-1 reference stock solution, respectively. Dissolve and dilute to the mark with acetonitrile and shake well. Prepare three replicates for each concentration.

[0149] Accurately measure the solvent (acetonitrile), the reference solution, and the recovery solutions at three concentrations, and inject them separately into the gas chromatograph. Record the chromatograms, calculate the amount of each known impurity by peak area using the external standard method for impurity references, and calculate the recovery rate. The results are shown in Table 18.

[0150] Table 18 Recovery rates of known impurities

[0151] Validation results: Within the three concentration ranges, the average recovery rates of impurity ii-3 in each accuracy solution were 102.3%, ii-9 96.0%, ii-2 103.8%, ii-4 102.4%, ii-5 96.6%, ii-8 105.0%, ii-10 98.9%, and ii-1 96.7%. The average recovery rates of all known impurities were between 90% and 108%, indicating that the method has high accuracy.

[0152] 8. Robustness Test: Under conditions where the carrier gas flow rate (1.8 ml / min ~ 2.2 ml / min), initial column temperature (45℃ ~ 60℃), split ratio (15:1 ~ 25:1), injection port temperature (255℃ ~ 265℃), and detector temperature (315℃ ~ 325℃) were varied, and different batches of the same brand, specification, and model of chromatographic column were used, the RSD of the retention time of each impurity under all conditions was less than 1%, the RSD of the peak area was less than 10%, the signal-to-noise ratio of the main peak height in the reference solution chromatogram was greater than 100, and the resolution between each impurity and adjacent peaks in the spiked test solution was greater than 1.5 (except for impurities ii-9 and ii-10). The detection results of each impurity were consistent with those under the standard conditions, indicating good method robustness.

[0153] Comparative Example 1: Different Chromatographic Columns A DB-35 column was used, and other chromatographic conditions were the same as in Example 1. The test solution was prepared (refer to the specificity section for preparation) and analyzed. The results are shown in [Figure 1]. Figure 4 .

[0154] As shown in the figure, the number of known impurities has decreased significantly. It is speculated that the known impurities overlap with the solvent or the main peak. Therefore, this type of chromatographic column is not suitable for the detection of related substances in this product.

[0155] Comparative Example 2: Different Gradient Procedures The following heating program and temperature conditions were used, with other conditions being the same as in Example 1: the initial temperature was 50°C, maintained for 3 minutes, then increased to 200°C at a rate of 10°C per minute, and maintained for 5 minutes. Injector temperature: 180°C, detector temperature: 250°C; carrier gas flow rate: 3 ml / min, split ratio: 5:1.

[0156] Under these conditions, the spiked test solution (containing 20 mg / ml of the main component and 40 µg / ml of each of the known impurities ii-1, ii-2, ii-3, ii-4, ii-5, ii-8, ii-9, and ii-10) was tested. The test results are shown in [Figure number missing]. Figure 5 One impurity peak is missing. Based on the relative positions of the chromatographic peaks, it is inferred that impurity ii-9 / 10 overlaps with the main peak under these chromatographic conditions. Therefore, this method cannot be used for the detection of related substances in this product.

[0157] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine, characterized in that, The gas chromatography method is used to detect related substances, and the chromatographic conditions of the gas chromatography method are as follows: A capillary column with 100% dimethylpolysiloxane as the stationary phase was used as the chromatographic column; the column flow rate was 1.8~2.2 ml per minute; The split ratio is 15:1 to 25:1; The heating program is as follows: the initial temperature is 45~60℃, maintained for 3 minutes, then increased to 160℃ at a rate of 8℃ per minute, maintained for 8 minutes, and then increased to 300℃ at a rate of 15℃ per minute, maintained for 10 minutes. The injection port temperature is 255~265℃; the detector is a flame ionization detector with a detector temperature of 315~325℃. The sample injection liner uses a glass frit liner.

2. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, The chromatographic column was an Agilent DB-1, 0.32 mm × 30 m, 0.5 μm.

3. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, Related substances of (3S,5S)-3-Boc-amino-5-methylpiperidine include: (2S,4S)-1,5-diamino-4-methyl-1,5-dioxolane-2-yl)carbamate, (1S,3S)-1,3-dicyanobutyl)carbamate, pyridine, {[(3S,5S)-1-benzyl-5-methylhexahydropyridin-3-yl]amino}methane-2-methylpropyl-2-yl ester, benzylamine, (S)-3-Boc-aminopiperidine, (3S,5R)-5-methylpiperidine-3-yl)carbamate, (3R,5S)-5-methylpiperidine-3-yl)carbamate.

4. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, It also includes sample preparation, with the test solution prepared in acetonitrile; the concentration of the test solution is 20±2 mg / ml; and the concentration of the control solution is 40±4 μg / ml.

5. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, The sample must not be stored in a glass container for more than 5 minutes; the container includes a sample vial. The sample in particular is a self-control solution.

6. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, A glass dropper was used during sample preparation.

7. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, The initial temperature is 50℃, maintained for 3 minutes, then increased to 160℃ at a rate of 8℃ per minute, maintained for 8 minutes, and then increased to 300℃ at a rate of 15℃ per minute, maintained for 10 minutes.

8. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, The injection port temperature is 260℃.

9. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, The detector is a flame ionization detector.

10. The method for detecting related substances in (3S,5S)-3-Boc-amino-5-methylpiperidine according to claim 1, characterized in that, The detector temperature is 320℃.

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