Method for detecting residual solvent 3, 5-dimethylpiperidine in tilmicosin bulk drug

Abstract

The invention belongs to the technical field of drug solvent residue detection, and particularly relates to a method for detecting a residual solvent 3, 5-dimethylpiperidine in a tilmicosin bulk drug. The method comprises the following steps: preparing a reference solution and a test solution, determining the content of the residual solvent 3, 5-dimethylpiperidine in the tilmicosin bulk drug by adopting gas chromatography-headspace sampling, and calculating the residual quantity according to an external standard method. The detection method is simple, accurate, good in repeatability and high in sensitivity; the method is suitable for daily detection of 3, 5-dimethylpiperidine serving as a tilmicosin raw material medicine, and the medication safety of tilmicosin is ensured by controlling the residual solvent amount in veterinary medicine tilmicosin, so that infectious diseases of animals such as pigs and chickens can be effectively and safely prevented and treated. According to the method, the blank of the method for detecting the residual solvent 3, 5-dimethylpiperidine in the tilmicosin is filled, the quality of the raw material medicine is effectively improved, and the clinical medication risk is reduced.

Description

Technical Field

[0001] This invention belongs to the field of drug solvent residue detection technology, specifically relating to a method for detecting residual solvent 3,5-dimethylpiperidine in tilmicosin active pharmaceutical ingredient. Background Technology

[0002] Tilmicosin, a derivative of tylosin, is a macrolide antibiotic for animal use. It boasts numerous advantages, including strong antibacterial activity, high safety, low resistance, low residue, and few adverse reactions. Tilmicosin has a similar molecular structure and antibacterial spectrum to tylosin, exhibiting strong antibacterial activity against mycoplasma, mycoplasma, spirochetes, most Gram-positive bacteria, and some Gram-negative bacteria. Due to its good cell permeability, it also has a good inhibitory effect on intracellular parasites. Clinically, it is mainly used for the prevention and treatment of acute and chronic respiratory and digestive system diseases in animals. Tilmicosin can dose-dependently inhibit PRRSV replication, showing inhibitory effects on both PRRSV1 and PRRSV2 types, and can reduce secondary infections by other pathogens, providing excellent protection for pig health.

[0003] These antibiotics are mainly produced through microbial fermentation or semi-synthetic processes, resulting in complex compositions. During fermentation, microorganisms may produce various substances and organic compounds. Organic solvents are commonly used in the production process to crystallize and purify crude tilmicosin. If process parameters are not strictly controlled, residual organic solvents may remain in the finished product. When the level of residual solvents in the drug exceeds safe limits, it may pose potential hazards to animals, humans, or the environment. Market sampling tests have revealed that some raw materials have a strong, irritating odor, and workers who came into contact with them experienced allergic reactions such as itching and redness, suggesting potential solvent residue issues.

[0004] In the synthesis of tilmicosin, solvents such as butyl peracetate and 3,5-dimethylpiperidine are used. The residue of these solvents not only reduces product quality but also affects drug safety. However, there are no reports in China on the detection of residual 3,5-dimethylpiperidine organic solvents. Therefore, establishing a detection method for 3,5-dimethylpiperidine is of great significance for ensuring the safety of tilmicosin use.

[0005] Currently, there is no readily available detection and analysis method for 3,5-dimethylpiperidine residues. This situation results in the lack of a unified standard for the drug, which greatly hinders its application and promotion. Therefore, in order to effectively control the quality of tilmicosin raw materials and ensure the safety of clinical use, it is essential to establish an effective and stable method for detecting solvent residues, which aims to provide a reference for the quality control of this raw material. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to propose a method for detecting the residual solvent 3,5-dimethylpiperidine in tilmicosin active pharmaceutical ingredient, so as to provide a reference for the quality control of the active pharmaceutical ingredient and promote the application and promotion of the drug.

[0007] Based on this, the technical solution of the present invention is as follows: A method for detecting residual solvent 3,5-dimethylpiperidine in tilmicosin active pharmaceutical ingredient, employing gas chromatography-headspace sampling, includes the following steps: (1) Preparation of test solution: Accurately weigh tilmicosin raw material, dissolve it in dimethyl sulfoxide, place it in a headspace bottle, seal it, and the solution is obtained; (2) Preparation of reference solutions: Accurately weigh 3,5-dimethylpiperidine reference standard, dissolve and dilute it with dimethyl sulfoxide to prepare a series of reference solutions of different concentrations; (3) Chromatographic detection: The test solution from step (1) and the reference solution from step (2) are respectively injected into the gas chromatograph via headspace injection for analysis, and the chromatograms are recorded. (4) Content calculation: Using the external standard method, a standard curve is established with the concentration of the reference solution in step (2) as the abscissa and the chromatographic peak area as the ordinate. Based on the chromatographic peak area of ​​the test solution measured in step (3), the content of 3,5-dimethylpiperidine in the test solution is calculated.

[0008] Furthermore, in step (3), the chromatographic column used for chromatographic detection is selected as a capillary column with a stationary phase of 6% cyanopropylbenzene + 94% dimethylsiloxane, polyethylene glycol or nitroterephthalic acid modified polyethylene glycol, or a gas capillary column with equivalent performance (preferably a capillary column with a stationary phase of 6% cyanopropylbenzene + 94% dimethylsiloxane).

[0009] Furthermore, the capillary column with the stationary phase of 6% cyanopropylbenzene + 94% dimethylsiloxane is a DB-624 column, 30m × 0.53mm × 1.8μm; The capillary column with polyethylene glycol as the stationary phase is a DB-WAX column, 30m × 0.32mm × 0.25μm; The capillary column with nitroterephthalic acid-modified polyethylene glycol as the stationary phase was a DB-FFAP column, 30m×0.32mm×0.5μm.

[0010] Furthermore, the chromatographic column used for chromatographic detection in step (3) is preferably a DB-624 column, 30m×0.53mm×1.8μm.

[0011] Furthermore, the chromatographic detection conditions in step (3) are as follows: Detector: Hydrogen flame ionization detector; Temperature rise program: Initial column temperature 58-62℃ (preferably 60℃) held for 2 min, then increased to 240℃ at a rate of 8-12℃ / min (preferably 10℃ / min) and held for 5 min; Inlet temperature: 240℃; Detector temperature: 280℃; Headspace flask equilibration temperature: 80℃, equilibration time: 30 minutes; Carrier gas: Nitrogen; Carrier gas flow rate: 1.0~1.6 mL / min (preferably 1.3 mL / min).

[0012] Furthermore, the injection volume for chromatographic detection in step (3) is 0.5~2μL (preferably 1μL).

[0013] Furthermore, in step (3), the injection split ratio for chromatographic detection is 5 to 20:1 (preferably 10:1).

[0014] Furthermore, the series of reference solutions described in step (2) have a concentration range of 0.0003 mg / mL to 0.8 mg / mL.

[0015] This invention also provides the application of the above-mentioned detection method in the quality control of tilmicosin active pharmaceutical ingredient or preparation.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention establishes a gas chromatography-headspace sampling method for determining the content of residual solvent 3,5-dimethylpiperidine in tilmicosin active pharmaceutical ingredient (API). The method is simple, accurate, reproducible, and highly sensitive. It is suitable for the routine detection of residual solvents in tilmicosin API, filling the gap in the detection method of 3,5-dimethylpiperidine in tilmicosin, and helping to improve the quality of API more effectively and reduce the risks of clinical medication. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is the chromatogram of the blank solution in Example 1 of the present invention; Figure 2 This is the chromatogram of the reference solution in Example 1 of the present invention; Figure 3 This is the chromatogram of the test solution in Example 1 of the present invention; Figure 4 This is a linear relationship diagram of 3,5-dimethylpiperidine in Example 1 of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] The term "active pharmaceutical ingredient" refers to a drug that consists of the main active pharmaceutical ingredient and impurities in controlled amounts.

[0021] The term "preparation" refers to a specific type of drug formulation (dosage form) prepared in accordance with standards approved by the pharmacopoeia or drug regulatory authorities, consisting of a main active pharmaceutical ingredient and impurities and / or excipients in controlled amounts, to meet clinical treatment or prevention needs.

[0022] The term "residual solvent" refers to any solvent substance that affects the purity of a drug.

[0023] The term "external standard method" refers to a method that uses a pure sample of the analyte as a reference substance and compares the response signals of the reference substance and the analyte in the sample for quantification.

[0024] Without violating common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention. For experimental procedures and methods not described in detail in the embodiments, standard operating procedures are used, such as the operating methods for solution preparation and system adaptability testing.

[0025] Unless otherwise specified, all reagents and raw materials used in the following examples are commercially available.

[0026] Example 1: Method for detecting residual solvent 3,5-dimethylpiperidine in tilmicosin active pharmaceutical ingredient. 1. Instruments and reagents Instrument: Agilent 8860 gas chromatograph (equipped with a flame ionization detector, FID); Nitrogen-hydrogen-air generator (Beijing Zhonghuipu Analytical Technology Research Institute); Open LBACDS Chem Station Edition workstation; Mettler ME204 electronic analytical balance (Germany).

[0027] Reagents: Tilmicosin raw material (Hubei Zhongmu Anda Pharmaceutical Co., Ltd.); 3,5-dimethylpiperidine, dimethyl sulfoxide (both chromatographic grade).

[0028] 2. Chromatographic conditions Chromatographic column: DB-624 capillary column, 30m × 0.53mm × 1.8μm.

[0029] Programmed temperature ramp: Initial column temperature is 60℃, hold for 2 min, then increase to 240℃ at a rate of 10℃ / min, hold for 5 min; Inlet temperature: 240℃; Detector temperature: 280℃; Headspace flask equilibration temperature: 80℃, equilibration time: 30min; Carrier gas: Nitrogen; Flow rate: 1.3 ml / min.

[0030] Split ratio: 10:1.

[0031] 3. Solution preparation Blank solution (blank solvent): dimethyl sulfoxide; Reference stock solution: Accurately weigh 500.00 mg of 3,5-dimethylpiperidine, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, shake well, and you will get a reference stock solution with a concentration of 5 mg / mL. Reference solution: Accurately measure 5 ml of the reference stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with dimethyl sulfoxide to obtain the reference solution.

[0032] Test solution: Accurately weigh 1.0269 g of tilmicosin raw material, place it in a headspace vial, accurately add 2 mL of dimethyl sulfoxide to dissolve it, seal the vial, and the test solution is obtained.

[0033] 4. Methodological Validation 4.1 Specificity test Take 1 μL each of the blank solution, reference solution, and test solution, and inject them into the gas chromatograph via headspace injection for analysis. Record the chromatograms. The results are as follows: Figures 1-3 As shown.

[0034] The results showed that under these chromatographic conditions, 3,5-dimethylpiperidine eluted, and all component peaks were baseline separated with a resolution greater than 1.5. The solvent did not interfere with the sample determination, and other impurity peaks were well separated from the main peak.

[0035] 4.2 Limit of Quantitation and Limit of Detection Test Accurately measure and serially dilute the reference standard to prepare a solution with a signal-to-noise ratio (S / N) of approximately 10, which will serve as the limit of quantitation solution.

[0036] Accurately measure 3 ml of the limit of quantitation solution and place it in a 10 ml volumetric flask. Dilute to the mark with dimethyl sulfoxide and shake well to obtain the limit of detection solution.

[0037] Solutions with limits of quantitation and limits of detection were injected into a gas chromatograph via headspace sampling for analysis. Each solution was measured six times consecutively, and the average value was taken. The results are shown in Table 1.

[0038] Table 1: Results of tests for limit of quantitation and limit of detection The above experimental results show that the peak area RSD of 3,5-dimethylpiperidine in 6 determinations was 1.1%, the limit of quantitation concentration was 0.323 μg / ml, and the limit of detection concentration was 0.106 μg / ml.

[0039] 4.3 Linearity Test Limit of Quantification Solution: Accurately measure the reference standard and dilute it stepwise to prepare a solution with a signal-to-noise ratio (S / N) of approximately 10, which is used as the limit of quantification solution.

[0040] 60% linear solution: Accurately measure 1.5 ml of the reference stock solution, place it in a 25 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well.

[0041] 80% linear solution: Accurately measure 2.0 ml of the reference stock solution, place it in a 25 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well.

[0042] 100% linear solution: Accurately measure 2.5 ml of the reference stock solution, place it in a 25 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well.

[0043] 120% linear solution: Accurately measure 3.0 ml of the reference stock solution, place it in a 25 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well.

[0044] 160% linear solution: Accurately measure 4.0 ml of the reference stock solution, place it in a 25 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well.

[0045] Accurately pipette the above solutions and inject them according to the above chromatographic conditions. Record the peak area and perform linear regression with peak area (y) as the ordinate and concentration (x, mg / mL) as the abscissa. The results are shown in Table 2.

[0046] Table 2: Results of the linear experiment See attached linear relationship diagram. Figure 4 .

[0047] Experimental results: 3,5-Dimethylpiperidine showed good linearity in the concentration range of 0.0003-0.8 mg / ml.

[0048] 4.4 Accuracy Test Preparation of standard addition solutions: Accurately measure 0.8 ml, 1.0 ml and 1.2 ml of the reference stock solution and place them in a 50 ml volumetric flask. Dilute with dimethyl sulfoxide to the mark and shake well to obtain standard addition solutions (1), (2) and (3). Preparation of spiked solution: Accurately weigh about 1.0g of tilmicosin raw material, make 9 portions, divide into 3 groups (3 portions in each group), place them in empty top bottles, accurately add 5.0ml of the above standard spiked solution to each bottle, and seal the bottle.

[0049] The spiked solution was injected into a gas chromatograph via headspace injection for analysis, and the chromatogram was recorded. The ratio of the measured amount to the added amount is the recovery rate (the same below). The results are shown in Table 3.

[0050] Table 3: Accuracy Test Results Note: The measured amount (mg) was calculated using the external standard method and has been included in the volume of the spiked solution (5.0 mL), and the background value has been deducted.

[0051] Experimental results: The average recovery rate of 3,5-dimethylpiperidine was 100.16%, and the RSD was 1.08%; the recovery rate results all met the requirements.

[0052] 4.5 Precision Test Take 5 ml of the reference solution and place it in a 20 ml headspace vial. Seal the vial and inject it into the gas chromatograph for analysis. Repeat the injection 6 times and record the chromatograms. Prepare 6 spiked solutions 2-1 and determine their content. Calculate the RSD of the recovery rate (%) and examine the precision of this method. The results are shown in Table 4.

[0053] Table 4: Precision Test Results Experimental results: The RSD (%) of the recovery of 3,5-dimethylpiperidine was 0.25%, indicating good sample repeatability.

[0054] 4.6 Solution stability test The reference solution was placed at room temperature for 0, 2, 4, 8, 12, and 24 hours. The reference solution at different placement times was then injected into the gas chromatograph via headspace for analysis. The chromatograms were recorded and the relative content (%) was calculated (the percentage of the measured amount relative to the added amount, the same below). The results are shown in Table 5.

[0055] Table 5: Results of Solution Stability Test Note: The relative content (%) is the percentage of the measured amount relative to the amount added, and the same applies below.

[0056] The experimental results showed that the RSD of the recovery rate of 3,5-dimethylpiperidine within 24 h at room temperature was 1.13%, indicating that the reference solution had good stability within 24 h at room temperature.

[0057] In summary, this embodiment establishes a method for determining the content of residual solvent 3,5-dimethylpiperidine in tilmicosin active pharmaceutical ingredient (API) using gas chromatography-headspace sampling. The method is simple, accurate, and highly sensitive, meeting the requirements for residual solvent detection. It is suitable for routine detection of residual solvents in tilmicosin API and provides a reference and basis for revising the residual solvent inspection item in the product's quality standard, effectively improving the quality of API and reducing the risks of clinical medication.

[0058] 4.7 Testing of the test sample The test solution was injected into the gas chromatograph via headspace sampling for analysis, and the chromatogram was recorded as follows: Figure 3 The test results are shown in Table 6.

[0059] Table 6 Test Results of the Samples The mass percentage of residual solvent 3,5-dimethylpiperidine in the test sample tilmicosin raw material was 0.0791%.

[0060] Example 2: Investigation of the Influence of Carrier Gas Velocity This embodiment is identical to the conditions in Example 1 except for the carrier gas flow rate. The reference solution was analyzed by headspace injection into a gas chromatograph. The chromatographic retention behavior was investigated at carrier gas flow rates of 1.1 mL / min, 1.3 mL / min, and 1.5 mL / min. The main peak retention time, tailing factor, and theoretical plate number were used as evaluation indicators. The relative content (%) of the residual solvent 3,5-dimethylpiperidine was calculated using the external standard method. The results are shown in Table 7.

[0061] Table 7: Test results at different carrier gas velocities As shown in Table 7, the retention time of the main peak, tailing factor, theoretical plate number and recovery rate meet the requirements within the carrier gas flow rate range of 1.1 mL / min to 1.5 mL / min.

[0062] Example 3: Investigation of the Influence of Initial Column Temperature This embodiment only changed the initial column temperature; all other conditions were the same as in Example 1. The reference solution was injected into the gas chromatograph via headspace analysis to investigate the chromatographic retention behavior at initial column temperatures of 58℃, 60℃, and 62℃. The main peak retention time, tailing factor, and theoretical plate number were used as evaluation indicators. The relative content (%) of the residual solvent 3,5-dimethylpiperidine was calculated using the external standard method. The experimental results are shown in Table 8.

[0063] Table 8: Test results at different initial column temperatures As shown in Table 8, within the initial column temperature range of 58℃ to 62℃, the retention time of the main peak, the tailing factor, the number of theoretical plates, and the recovery rate meet the requirements.

[0064] Example 4: Investigation of the effect of heating rate Except for the heating rate, the conditions in this embodiment are the same as in Example 1. The reference solution was analyzed by headspace injection into a gas chromatograph. The chromatographic retention behavior under heating rates of 8℃ / min, 10℃ / min, and 12℃ / min was investigated. The main peak retention time, tailing factor, and theoretical plate number were used as evaluation indicators. The relative content (%) of the residual solvent 3,5-dimethylpiperidine was calculated using the external standard method. The experimental results are shown in Table 9.

[0065] Table 9: Test Results at Different Heating Rates As shown in Table 9, within the heating rate range of 8℃ / min to 12℃ / min, the retention time of the main peak, the tailing factor, the number of theoretical plates, and the recovery rate meet the requirements.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting residual solvent 3,5-dimethylpiperidine in tilmicosin active pharmaceutical ingredient, characterized in that, The gas chromatography-headspace sampling method was employed, and included the following steps: (1) Preparation of test solution: Accurately weigh tilmicosin raw material, dissolve it in dimethyl sulfoxide, place it in a headspace bottle, seal it, and the solution is obtained; (2) Preparation of reference solutions: Accurately weigh 3,5-dimethylpiperidine reference standard, dissolve and dilute it with dimethyl sulfoxide to prepare a series of reference solutions of different concentrations; (3) Chromatographic detection: The test solution from step (1) and the reference solution from step (2) are respectively injected into the gas chromatograph via headspace injection for analysis, and the chromatograms are recorded. (4) Content calculation: Using the external standard method, a standard curve is established with the concentration of the reference solution in step (2) as the abscissa and the chromatographic peak area as the ordinate. Based on the chromatographic peak area of ​​the test solution measured in step (3), the content of 3,5-dimethylpiperidine in the test solution is calculated.

2. The detection method according to claim 1, characterized in that, In step (3), the chromatographic column selected for chromatographic detection is a capillary column with a stationary phase of 6% cyanopropylbenzene + 94% dimethylsiloxane, polyethylene glycol or nitroterephthalic acid modified polyethylene glycol, or a gas capillary column with equivalent performance.

3. The detection method according to claim 2, characterized in that, The capillary column with the stationary phase of 6% cyanopropylbenzene + 94% dimethylsiloxane is a DB-624 column, 30m × 0.53mm × 1.8μm; The capillary column with polyethylene glycol as the stationary phase is a DB-WAX column, 30m × 0.32mm × 0.25μm; The capillary column with nitroterephthalic acid-modified polyethylene glycol as the stationary phase was a DB-FFAP column, 30m×0.32mm×0.5μm.

4. The detection method according to claim 1, characterized in that, The chromatographic detection conditions in step (3) are as follows: Detector: Hydrogen flame ionization detector; Temperature program: Initial column temperature 58-62℃, hold for 2 min, then increase to 240℃ at a rate of 8-12℃ / min, hold for 5 min; Inlet temperature: 240℃; Detector temperature: 280℃; Headspace flask equilibration temperature: 80℃, equilibration time: 30 minutes; Carrier gas: Nitrogen; Carrier gas flow rate: 1.0~1.6 mL / min.

5. The detection method according to claim 1, characterized in that, The injection volume for chromatographic detection in step (3) is 0.5~2μL.

6. The detection method according to claim 1, characterized in that, In step (3), the injection split ratio for chromatographic detection is 5~20:

1.

7. The detection method according to claim 1, characterized in that, The series of reference solutions described in step (2) have a concentration range of 0.0003 mg / mL to 0.8 mg / mL.

8. The application of the detection method as described in any one of claims 1-7 in the quality control of tilmicosin active pharmaceutical ingredient or preparation.

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