Topiramate cleaning chemical residue detection method and kit

Abstract

The invention provides a topiramate cleaning chemical residue detection method and kit.The method comprises the steps that sampling is conducted in a specified area to obtain a sample, and the sampling step comprises wiping sampling and / or washing sampling; the method comprises the following steps: adding a mixed extraction solvent into a sample for extraction, filtering an extraction solution, adjusting the extraction solution to alkalescence, and adding a derivatization reagent for reaction to obtain a purified solution to be detected; the purified liquid to be detected is injected into a chromatographic detection system for quantitative detection, a detection result is output, and the detection step comprises chromatographic separation and signal response analysis. Accurate detection and limit judgment on topiramate residues on the surface of equipment are realized through standard sampling, extraction purification and derivatization enhanced response and in combination with chromatographic quantitative analysis, so that collinear production pollution and cross contamination risks of medicines are effectively controlled.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a method and kit for detecting topiramate cleaning chemical residues. Background Technology

[0002] Topiramate is a novel antiepileptic drug containing an aminosulfonate ester structure. Common dosage forms are film-coated tablets and hard capsules. Topiramate oral solutions utilize organic solubilizers to improve small intestinal permeability, enhancing bioavailability and making them suitable for patients with difficulty swallowing tablets or capsules, significantly improving patient compliance. During drug manufacturing, the active ingredient may adsorb onto the inner surfaces of production equipment (such as dispensing tanks and pipelines). According to the mandatory requirements for cleaning validation in the Good Manufacturing Practice (GMP) for pharmaceuticals, sensitive and reliable detection methods must be established to confirm that the residual amount of topiramate on the equipment surface after cleaning is below the predetermined safety limit, thereby preventing cross-contamination between drugs and ensuring patient safety. Topiramate oral solution is available in 25 mg / ml form. As a highly polar drug ingredient containing an aminosulfonate structure, the residual concentration of topiramate when produced on the same production line as other products is generally in the microgram per milliliter range. After cleaning, the residual level is usually extremely low and is accompanied by interference from cleaning agents, matrix impurities, etc., which leads to weak signal response, unstable recovery rate, and easy to miss or misjudge when directly detected. As a result, it is difficult to meet the mandatory requirements of sensitivity and reliability for cleaning validation. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method and kit for detecting chemical residues of topiramate after cleaning, which aims to solve the problem that topiramate may still remain in trace form after cleaning of production equipment and is difficult to be accurately identified by conventional quantitative detection.

[0004] To solve the above-mentioned technical problems, the present invention is implemented as follows: The present invention proposes a method for detecting topiramate cleaning chemical residues, the steps of which include: S1. Take samples from a specified area to obtain samples, wherein the sampling steps include wiping sampling and / or rinsing sampling; S2. Add a mixed extraction solvent to the sample for extraction, filter the extract and adjust it to weak alkalinity, then add a derivatization reagent to react and obtain the purified solution to be tested. S3. Inject the purified solution to be tested into the chromatographic detection system for quantitative detection and output the detection results. The detection steps include chromatographic separation and signal response analysis.

[0005] In some embodiments of the present invention, step S2 includes: S2.1 Place the sample in a stoppered centrifuge tube, use a pipette to precisely add the mixed extraction solvent for extraction, vortex for 30-60 seconds after adding, and then place it in an ultrasonic cleaner for ultrasonic extraction. The ultrasonic time is controlled to be 8-15 minutes. S2.2 After ultrasonic extraction, the extract is filtered through a 0.45-micron organic microporous membrane, and the filtrate is collected and used as the purified extract. S2.3. Accurately measure the purified extract, add alkaline solvent to adjust the system to weak alkalinity, then add derivatization reagent solution and mix well, then place in a 60℃ constant temperature water bath to react in the dark for 25~35 min. S2.4 After the derivatization reaction is completed, remove the sample and cool it to room temperature. Then, dilute it to the mark with phosphate buffer solution and shake well to obtain the purified solution to be tested.

[0006] In some embodiments of the present invention, in step S2, the mixed extraction solvent includes at least one of acetonitrile-water mixed solvent, methanol-water mixed solvent, ethanol-water mixed solvent, isopropanol-water mixed solvent, and ethyl acetate-water mixed solvent; the alkaline solvent includes at least one of sodium bicarbonate solution, sodium carbonate solution, borate buffer, disodium hydrogen phosphate solution, and ammonia water; and the derivatizing reagent includes at least one of 2,4-dinitrofluorobenzene, p-nitrobenzenesulfonyl chloride, dansyl chloride, and 9-fluorenemethoxycarbonyl chloride.

[0007] In some embodiments of the present invention, step S1 includes: S1.1 For flat, wipeable areas, use a dust-free cotton swab or polyester fiber wiping cloth for wiping and sampling. Before wiping, pre-wet the wiping material. The solvent used for pre-wetting should be the same as or compatible with the mixed extraction solvent. For areas that cannot be effectively wiped, use a rinsing sampling method. Use a fixed volume of rinsing solution to rinse repeatedly and recover the rinsing solution as the rinsing sample. The rinsing solution should be the same as or compatible with the mixed extraction solvent. S1.2. A blank control sample shall be set up simultaneously in each sampling batch. The blank control sample shall include pre-wetting wiping material that has not been in contact with the equipment surface or rinsing liquid that has not been rinsed by the equipment.

[0008] In some embodiments of the present invention, step S3 includes: S3.1 The purified solution to be tested is used for chromatographic detection. The chromatographic detection system is a high performance liquid chromatograph equipped with an ultraviolet detector. The chromatographic separation uses a reversed phase chromatographic column as the analytical column, and the column temperature is controlled at 35~45℃. S3.2 The mobile phase in the high performance liquid chromatograph includes an aqueous buffer and an organic solvent. An autosampler is used for injection, and the injection volume is controlled at 20~100μL. S3.3. Prepare a reference solution of topiramate derivative. The reference solution is prepared according to the same derivatization process as the test purification solution, and the quantitative calculation is performed based on the correspondence between peak area and concentration to output the residual concentration of topiramate in the sample. S3.4. The detected topiramate residual concentration is converted into residual amount per unit area based on the sampling area, and compared with the preset cleaning limit to output the cleaning qualification result.

[0009] In some embodiments of the present invention, in step S3, the aqueous buffer includes at least one of sodium acetate buffer, phosphate buffer, ammonium acetate buffer, ammonium formate buffer, and citrate buffer, and the organic solvent includes at least one of acetonitrile, methanol, ethanol, isopropanol, and tetrahydrofuran.

[0010] In some embodiments of the present invention, the formula for calculating the residual amount per unit area in step S3 is as follows: In the formula, m represents the residual amount per unit area, with units of μg / cm². 2 ; The peak area is the peak area measured after derivatization of the test solution. The concentration of topiramate in the reference solution is expressed in μg / ml. The peak area is measured after derivatization of the reference solution; S represents the total volume of the sample after dilution during extraction, in ml; S represents the sampling area, in cm². 2 ; The measured residue per unit area is corrected using the sampling recovery rate, and the unit is μg / cm². 2 N represents the sampling recovery rate.

[0011] In some embodiments of the present invention, in step S3, the preset cleanliness limit is 0.22 μg / cm³. 2 If the residual amount per unit area is less than or equal to the preset cleaning limit, the result of cleaning is qualified; if the residual amount per unit area is greater than the preset cleaning limit, the result of cleaning is unqualified.

[0012] This invention proposes a kit for implementing a method for detecting topiramate clean chemical residues as described above. The kit contains a mixed extraction solvent and a derivatization reagent.

[0013] The advantages of the topiramate cleaning chemical residue detection method and kit in this invention compared with the prior art are as follows: Step S1 involves obtaining residual samples from the equipment surface within a specified area using wiping and / or rinsing sampling, ensuring that residues in planar areas and areas that cannot be effectively wiped are covered and collected. Step S2 involves extracting the sample by adding a mixed extraction solvent to fully dissolve the topiramate residue, filtering the extract to remove impurities, and adjusting the system to a weakly alkaline state before adding a derivatization reagent to react and form a derivative with a stronger signal response, thereby improving detection sensitivity and stability. Step S3 involves injecting the obtained purified solution into a chromatographic detection system, using chromatographic separation and signal response analysis to achieve quantitative detection of the topiramate derivative and output the detection results. This accurately confirms whether the amount of topiramate residue after equipment cleaning is below the predetermined safety limit, meets cleaning validation requirements, and prevents cross-contamination. Attached Figure Description

[0014] Figure 1 This is a schematic flowchart of a method for detecting chemical residues in topiramate cleaning products according to an embodiment of the present invention.

[0015] Figure 2 This is a chromatogram of the blank solution used in the precision test of this invention.

[0016] Figure 3 This is a chromatogram of the reference solution used in the precision test of this invention.

[0017] Figure 4 This is a chromatogram of the test solution used in the precision test of this invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] Please refer to Figure 1 This invention proposes a method for detecting topiramate cleaning chemical residues, the steps of which include: S1. Take samples from a specified area to obtain samples, wherein the sampling steps include wiping sampling and / or rinsing sampling.

[0020] Step S1 includes: S1.1 For wipeable flat areas, use a dust-free cotton swab or polyester fiber wiping cloth for wiping and sampling. Before wiping, pre-wet the wiping material. The solvent used for pre-wetting should be the same as or compatible with the mixed extraction solvent. For areas that cannot be effectively wiped, use a rinsing sampling method. Use a fixed volume of rinsing solution to rinse repeatedly and recover the rinsing solution as the rinsing sample. The rinsing solution should be the same as or compatible with the mixed extraction solvent.

[0021] Step S1.1 involves wiping the flat area with a clean cotton swab or polyester fiber cloth to collect samples. Before wiping, the wiping material is pre-wetted with a solvent that is consistent with or compatible with the mixed extraction solvent. This forms a micro-liquid film with dissolving capabilities on the surface of the wiping medium, which chemically enhances the dissolution and migration efficiency of topiramate residues. Topiramate is highly polar and tends to remain in a trace adsorbed state on the surface of stainless steel or polymer equipment after cleaning. The pre-wetting solvent, such as an acetonitrile-water system or a methanol-water system, can disrupt the weak interaction between the residue and the surface through polarity matching, making it easier for the wiping material to adsorb and transfer to the sample, thereby improving the recovery rate of residue per unit area. For complex structural areas that cannot be effectively wiped, a quantitative volume of rinsing solution is used for repeated rinsing, and the rinsing solution is recovered as a sample. This utilizes the wetting and dissolution effect of the liquid on areas such as grooves and pipe inner walls to achieve comprehensive collection of hidden residues, avoiding sampling blind spots caused by a single wiping method, and improving the overall sampling integrity and detection reliability.

[0022] S1.2. A blank control sample shall be set up simultaneously in each sampling batch. The blank control sample shall include pre-wetting wiping material that has not been in contact with the equipment surface or rinsing liquid that has not been rinsed by the equipment.

[0023] Step S1.2 involves simultaneously setting up blank control samples in each sampling batch. Blank control samples include pre-wetting wiping material that has not come into contact with the equipment surface or rinsing solution that has not been rinsed by the equipment. The technical effect is to establish a background baseline and contamination control mechanism for the sampling process. Since trace impurities or environmental introducers may exist in the pre-wetting solvent, wiping material, and sampling container, the absence of blank controls may lead to background interference peaks in chromatographic detection, thus affecting the quantitative accuracy of topiramate derivatives. Blank control samples can identify non-target responses from the extraction solvent, derivatization reagent, or wiping material itself, and correct these signals in subsequent calculations, ensuring that the detection results originate from actual equipment residue rather than system contamination. Furthermore, this step verifies the cleanliness and batch consistency of the sampling process, enabling the detection system to meet the traceability and repeatability requirements of cleanliness validation, thereby improving the compliance and reliability of the method in specific scenarios.

[0024] S2. Add a mixed extraction solvent to the sample for extraction, filter the extract and adjust it to weak alkalinity, then add a derivatization reagent to react and obtain the purified solution to be tested. Step S2 includes: S2.1 Place the sample in a stoppered centrifuge tube, use a pipette to precisely add the mixed extraction solvent for extraction, vortex for 30-60 seconds after addition, and then place it in an ultrasonic cleaner for ultrasonic extraction. The ultrasonic time is controlled to be 8-15 minutes.

[0025] Step S2.1 involves adding a mixed extraction solvent, such as an acetonitrile-water mixture, a methanol-water mixture, or an ethanol-water mixture, to a stoppered centrifuge tube to achieve efficient dissolution and preliminary extraction of topiramate cleaning residues. Acetonitrile solvents have strong polarity matching capabilities, effectively disrupting the adsorption between topiramate molecules and the equipment surface, allowing them to rapidly enter the liquid phase system. Vortex mixing enhances the contact between the solvent and the sample, reducing local concentration gradients, while the cavitation effect generated by the ultrasonic cleaner promotes the release of residues from the interior of the wiping material fibers or microporous structures, thereby significantly improving the extraction efficiency and sample homogeneity of topiramate.

[0026] S2.2 After ultrasonic extraction, the extract is filtered through a 0.45-micron organic microporous membrane, and the filtrate is collected as the purified extract.

[0027] Step S2.2 After extraction, particulate impurities are removed by filtration through an organic microporous membrane to avoid clogging of the chromatographic system and improve injection stability.

[0028] S2.3. Accurately measure the purified extract, add an alkaline solvent to adjust the system to a weakly alkaline state, then add the derivatization reagent solution and mix well. Then place it in a 60℃ constant temperature water bath and react in the dark for 25~35 minutes.

[0029] Step S2.3: Accurately measure the purified extract and add an alkaline solvent such as sodium bicarbonate solution, sodium carbonate solution, or borate buffer to adjust the system to a weakly alkaline environment, making the reactive sites in the topiramate molecule more readily derivatized and activated. Under weakly alkaline conditions, adding a dinitrohaloaromatic derivatizing reagent such as dinitrofluorobenzene, dinitrochlorobenzene, or dinitrobenzene derivatives allows for nucleophilic substitution reactions with the reactive groups in the topiramate molecule, generating derivatives with strong UV absorption characteristics. The light-protected water bath reaction avoids photodegradation of the derivatizing reagent, while the isothermal conditions ensure stable reaction kinetics, thus significantly improving the UV detection sensitivity and quantitative accuracy of topiramate.

[0030] S2.4 After the derivatization reaction is completed, remove the sample and cool it to room temperature. Then, dilute it to the mark with phosphate buffer solution and shake well to obtain the purified solution to be tested.

[0031] Step S2.4, cooling to room temperature after derivatization, terminates the reaction and stabilizes the derivative structure, preventing side reactions or degradation under high-temperature conditions. Subsequently, a phosphate buffer solution is used for volume adjustment, providing stable ionic strength and acid-base buffering capacity, ensuring structural stability of the derivatized product during detection and reducing peak drift. Shaking ensures uniform system concentration, resulting in the purified analyte. This step, through reaction termination and volume standardization, achieves comparability between different batches of samples, improving the repeatability of quantitative results and the reliability of cleaning residue determination.

[0032] In step S2, the mixed extraction solvent includes at least one of acetonitrile-water mixed solvent, methanol-water mixed solvent, ethanol-water mixed solvent, isopropanol-water mixed solvent, and ethyl acetate-water mixed solvent; the alkaline solvent includes at least one of sodium bicarbonate solution, sodium carbonate solution, borate buffer, disodium hydrogen phosphate solution, and ammonia water; and the derivatizing reagent includes at least one of 2,4-dinitrofluorobenzene, p-nitrobenzenesulfonyl chloride, dansyl chloride, and 9-fluorenemethoxycarbonyl chloride.

[0033] S3. Inject the purified solution to be tested into the chromatographic detection system for quantitative detection and output the detection results. The detection steps include chromatographic separation and signal response analysis. Step S3 includes: S3.1 The purified solution to be tested is used for chromatographic detection. The chromatographic detection system is a high performance liquid chromatograph equipped with an ultraviolet detector. The chromatographic separation uses a reversed phase chromatographic column as the analytical column, and the column temperature is controlled at 35~45℃.

[0034] Step S3.1 utilizes a high-performance liquid chromatograph equipped with a UV detector and a reversed-phase column as the analytical column to achieve effective chromatographic separation and signal detection of topiramate derivatives. The stationary phase inside the reversed-phase column has hydrophobic characteristics, enabling topiramate derivatives and residual impurities to exhibit different retention behaviors within the column, thus achieving separation based on elution time differences. Controlling the column temperature within the range of 35–45°C stabilizes the mobile phase viscosity and partition equilibrium, avoiding retention time drift and peak diffusion, improving detection repeatability and peak symmetry, and ensuring that topiramate derivatives still yield clear and identifiable characteristic peaks even in complex and clean matrices, thereby enhancing the reliability of quantitative analysis.

[0035] S3.2 The mobile phase in the high performance liquid chromatograph includes an aqueous buffer and an organic solvent. An autosampler is used for injection, and the injection volume is controlled at 20~100μL.

[0036] Step S3.2 involves adjusting the chromatographic elution capacity and optimizing the separation selectivity of topiramate derivatives by setting up a mobile phase system composed of an aqueous buffer and an organic solvent. The aqueous buffer, such as sodium acetate buffer, phosphate buffer, or ammonium acetate buffer, maintains the acid-base stability of the system, reduces dissociation fluctuations of the derivatives during separation, thereby improving peak shape and reducing tailing. The organic solvent, such as acetonitrile, methanol, or tetrahydrofuran, enhances the elution intensity of the mobile phase, allowing the target component to elute within a reasonable time range and effectively separate from impurity peaks. Using an autosampler and controlling the injection volume within the range of 20–100 μL ensures injection consistency, reduces human error, and makes the detection results more in line with the sensitivity and repeatability requirements of clean validation.

[0037] S3.3. Prepare a reference solution of topiramate derivative. The reference solution is prepared according to the same derivatization process as the test purification solution, and the quantitative calculation is performed based on the correspondence between peak area and concentration to output the residual concentration of topiramate in the sample.

[0038] Step S3.3 involves preparing a reference solution of topiramate derivatives to achieve accurate external standard quantification of topiramate residual concentration. The reference solution is prepared using the same derivatization process as the test solution, ensuring that the reference solution and the topiramate derivative in the sample have consistent chemical forms and UV response characteristics, thereby eliminating systematic bias caused by differences in derivatization efficiency. By establishing the correlation between peak area and concentration, the chromatographic signal can be directly converted into a residual concentration value, improving the accuracy and traceability of quantitative analysis, providing a quality control basis for the detection process, and meeting the standardized quantification requirements in cleaning validation.

[0039] S3.4. The detected topiramate residual concentration is converted into residual amount per unit area based on the sampling area, and compared with the preset cleaning limit to output the cleaning qualification result.

[0040] Step S3.4 converts the detected topiramate residual concentration into a residual amount per unit area by combining it with the sampling area, thus achieving a direct correspondence between the detection results and the equipment surface cleanliness limits. This conversion method transforms the concentration index in the solution into the residual level on the equipment surface, making the results more consistent with the actual needs of cleanliness validation management, which uses residual amount per unit area as the judgment criterion. Further comparison of the residual amount per unit area with the preset cleanliness limits directly outputs the cleanliness compliance judgment result, thereby realizing a closed-loop application of topiramate residual detection from experimental determination to release decision-making. This ensures that the risks of contamination and cross-contamination during co-production of pharmaceuticals are effectively controlled and guarantees the safety and compliance of different batches of pharmaceuticals.

[0041] In step S3, the aqueous buffer includes at least one of sodium acetate buffer, phosphate buffer, ammonium acetate buffer, ammonium formate buffer, and citrate buffer, and the organic solvent includes at least one of acetonitrile, methanol, ethanol, isopropanol, and tetrahydrofuran.

[0042] In step S3.2, the ultraviolet detection wavelength is set to 350nm to 370nm as the main detection wavelength.

[0043] Setting the UV detection wavelength to 350 nm to 370 nm as the main detection wavelength in step S3.2 significantly enhances selectivity. Topiramate technical molecule itself lacks strong UV absorption groups, resulting in a weak signal response during direct detection. However, after adding derivatizing reagents such as dinitrofluorobenzene, dinitrochlorobenzene, or dansyl chloride in step S2, topiramate undergoes a nucleophilic substitution reaction with the derivatizing reagent to generate derivatives containing dinitro aromatic or sulfonyl aromatic structures. These derivatives exhibit distinct characteristic absorption peaks in the 350 nm to 370 nm wavelength range. By selecting this wavelength range for detection, topiramate derivatives can obtain a stronger UV response signal, thereby significantly improving the detection sensitivity for trace residues.

[0044] Furthermore, the 350nm to 370nm wavelength range is a relatively high wavelength region where common cleaning agent residues, solvent background, and equipment material leachates have weak absorption. Therefore, using this main detection wavelength can effectively reduce baseline noise and impurity interference caused by low-wavelength background absorption, reduce the influence of co-elution peaks on the target peak, and make the chromatographic signal purer and more stable. This setting can improve the specificity and quantitative accuracy of topiramate cleaning residue detection, thereby more reliably meeting the requirements of cleaning validation for low-limit residue determination. This invention proposes a kit for implementing the above-described method for detecting topiramate cleaning chemical residues. The kit contains a mixed extraction solvent and a derivatization reagent. In this embodiment, 360nm is preferred. Under alkaline and heating conditions, the primary amino group in the topiramate molecule nucleophilically attacks the carbon atom at the para position of the fluorine atom in the 2,4-dinitrofluorobenzene molecule, replacing the fluorine atom and generating a stable dinitrophenyl derivative. This derivative molecule contains a large conjugated system, thus exhibiting strong ultraviolet absorption near the 360nm wavelength.

[0045] The formula for calculating the residual amount per unit area is: In the formula, m represents the residual amount per unit area, with units of μg / cm². 2 ; The peak area is the peak area measured after derivatization of the purified liquid to be tested. The concentration of topiramate in the reference solution is expressed in μg / mL. The peak area is measured after derivatization of the reference solution; S represents the total volume of the sample after dilution during extraction, in ml; S represents the sampling area, in cm². 2 ; The measured residue per unit area is corrected using the sampling recovery rate, and the unit is μg / cm². 2N represents the sampling recovery rate, calculated by coating or spraying a reference solution with a known topiramate concentration, followed by derivatization, and then calculating the ratio of the actual recovered mass to the theoretical spiked mass on the surface. The unit is dimensionless. This equation for calculating the residual amount per unit area unifies the conversion between the topiramate residual concentration in the test purified solution obtained from chromatographic detection and the sampling area, and further incorporates sampling recovery rate correction. This ensures that the detection results not only reflect the apparent concentration in the solution but also accurately characterize the actual residual level of topiramate per unit area on the equipment surface. This solves the systematic underestimation or overestimation problems caused by incomplete wiping transfer, solvent extraction loss, and insufficient derivatization conversion in cleaning validation scenarios. Simultaneously, a quantitative requirement for the stability of chromatographic separation conditions is proposed, making S3.2 a crucial step in ensuring the reliability of the correction calculation.

[0046] Example 1: In this example, the topiramate cleaning chemical residue was detected according to the method described in claim 1. First, in step S1, a sample of a specified area of ​​10cm × 10cm was taken from the inner surface of the equipment. For flat areas, a dust-free cotton swab was used for wiping and sampling. Before wiping, the sample was pre-wetted with a mixture of acetonitrile and water, wherein the volume ratio of acetonitrile to water was 50:50. For the bends in the pipeline, a rinsing sampling method was used. The sample was rinsed three times with 10mL of the acetonitrile-water mixture and the rinsing solution was collected as the sample. At the same time, a blank control sample was set up. The blank control sample was the extract from the pre-wetted cotton swab that did not come into contact with the equipment surface.

[0047] In step S2, a swab is placed in a stoppered centrifuge tube, and 20 mL of acetonitrile-water mixed solvent is added using a pipette for extraction. After vortexing for 45 s, the tube is ultrasonically extracted for 10 min in an ultrasonic cleaner. After ultrasonication, 2.0 g of anhydrous magnesium sulfate and 0.5 g of sodium chloride are added as salting-out components. After vortexing for 1 min, the tube is centrifuged at 6000 rpm for 4 min, and the supernatant is used as the enriched extract. The enriched extract is then transferred to a purification tube for dispersion solid-phase purification. The purification tube is pre-loaded with 200 mg of PSA adsorbent and 200 mg of C18 reversed-phase adsorbent, and 1.0 g of anhydrous sodium sulfate is added as a dehydrating agent. After vortexing for 60 s, the tube is centrifuged at 10000 rpm for 4 min, and the supernatant is used as the purified extract. The purified extract was filtered through a 0.45 μm organic microporous membrane. The filtrate was collected and adjusted to a weakly alkaline state with 0.5 mL of sodium bicarbonate solution. Then, 3.0 mL of 2,4-dinitrofluorobenzene solution was added as a derivatization reagent. After mixing, the mixture was placed in a 60 °C water bath in the dark for 30 min. After the reaction was completed, the solution was cooled to room temperature and brought to a final volume of 25 mL with phosphate buffer solution. The solution was then shaken well to obtain the purified solution to be tested.

[0048] In step S3, the purified solution to be tested is injected into a high-performance liquid chromatograph (HPLC) for detection. An ultraviolet (UV) detector is configured, and a reversed-phase column is used with the column temperature controlled at 40°C. The mobile phase consists of phosphate buffer and acetonitrile, and an autosampler is used for injection at a volume of 50 μL. The UV detection wavelength is set to 360 nm. A reference solution is prepared using the same derivatization procedure as the purified solution to be tested. The topiramate concentration in the reference solution is 1.1 μg / mL. The residual amount per unit area (m) is calculated using the external standard method, combining the sampling area and the final volume. The residual amount per unit area is then corrected using the recovery rate and compared with the preset cleanliness limit of 0.22 μg / cm². 2 Compare and output the judgment result.

[0049] Example 2: This example follows the same steps as Example 1, except that a methanol-water mixture is used as the extraction solvent, with a methanol-to-water volume ratio of 60:40. A surface desorption promoter is further added to the extraction solvent to enhance the desorption efficiency of topiramate on the stainless steel or polymer inner wall surface. The surface desorption promoter is Tween-80, added at 0.05 wt%, allowing topiramate residues to more fully enter the extraction system from the equipment surface. The salting-out components are 2.5 g of anhydrous magnesium sulfate and 0.8 g of sodium acetate. The purification adsorbent is 150 mg of graphitized carbon black and 250 mg of C18 reversed-phase adsorbent. The dehydrating agent is 0.8 g of anhydrous calcium chloride. Weak alkalinity is adjusted using 0.5 mL of sodium carbonate solution, and the derivatization reagent is 3.0 mL of dansyl chloride solution. The chromatographic mobile phase aqueous buffer is sodium acetate buffer, the organic solvent is methanol, the UV detection wavelength is set to 365 nm, and the injection volume is 80 μL. The remaining steps and conditions are consistent with Example 1.

[0050] Example 3: This example follows the same steps as Example 1, except that the mixed extraction solvent is an ethanol-water mixture with a volume ratio of 70:30. The salting-out components are 0.6g sodium chloride and 0.7g sodium citrate. After ultrasonic extraction, an ionic strength synergist is added to the centrifuge tube and vortexed for 30-60 seconds to enhance the enrichment and distribution of topiramate in the extraction system. The ionic strength synergist is potassium chloride, added at a rate of 0.10g / 20mL of extract. Then, sodium chloride and sodium citrate are added as in step S2.2 for salting-out layering. The purification adsorbent is 300mg of polymeric adsorption resin and 150mg of PSA adsorbent, and the dehydrating agent is 1.0g of anhydrous calcium phosphate. Weak alkalinity is adjusted using 0.5mL borate buffer, and the derivatization reagent is 3.0mL of 9-fluorenemethyloxycarbonyl chloride solution. The mobile phase aqueous buffer was citrate buffer, the organic phase solvent was acetonitrile, the ultraviolet detection wavelength was set to 370 nm, the injection volume was 60 μL, and the remaining steps and conditions were the same as in Example 1.

[0051] Comparative Example 1: Except for step S2.2, in which anhydrous magnesium sulfate and sodium chloride were not added to the salt-out components, the other steps of this comparative example were completely consistent with those of Example 1, that is, after ultrasonic extraction, the supernatant was directly centrifuged and purified.

[0052] Comparative Example 2: This comparative example is based on Example 1 with additional settings. Except for not using the formula for calculating the residual amount per unit area, the sampling, extraction and enrichment, derivatization, and high-performance liquid chromatography detection steps are completely consistent with Example 1. That is, the wiping and / or rinsing sampling is still performed within the specified area according to step S1. The acetonitrile-water mixed solvent extraction, salting-out layering, dispersion solid-phase purification, weak alkalinity adjustment, and 2,4-dinitrofluorobenzene derivatization reaction are performed according to step S2, and the volume is adjusted to obtain the purified solution to be tested. The test purified solution is then detected according to step S3 using a high-performance liquid chromatograph equipped with an ultraviolet detector, a reversed-phase column, column temperature controlled at 35 to 45°C, mobile phase composed of phosphate buffer and acetonitrile, autosampler injection with an injection volume of 20 to 100 μL, and ultraviolet detection wavelength in the range of 350 to 370 nm. The residual concentration Ccorr of the purified solution to be tested is calculated.

[0053] The relative deviation data for parallel determinations are derived from repeated determination experiments on the same sample. The specific steps are as follows: within 24 hours, the residual sample on the same sampling surface is subjected to no less than 3 parallel treatments, including the same ultrasonic extraction, salting-out layering, purification and dehydration, derivatization reaction and chromatographic detection, and then the relative standard deviation of the residual amount per unit area is calculated.

[0054] The data on the differences in retesting results by different operators came from personnel replication verification experiments. The experimental procedure was as follows: two or more operators independently completed sampling, extraction, derivatization, and chromatographic detection using the same equipment and at the same residue level, and then compared the differences in the final calculated residue per unit area.

[0055] Experimental Procedure for Sampling Recovery: The sampling recovery rate was determined using a manual spiked recovery verification method. First, a 10 cm × 10 cm stainless steel or polymer sample of the same material as the actual production equipment was selected. A known concentration of topiramate standard solution was uniformly added to the surface of the sample, ensuring the spiked amount per unit area was near the cleanliness limit. After the solvent evaporated naturally, a simulated residue layer was formed. Then, a wiping sampling was performed according to step S1 in the previous example, using the same solvent as in the corresponding example for pre-wetting the wiping material. After wiping, the wiping material was placed in a stoppered centrifuge tube, and extraction, salting-out, dispersion solid-phase purification, weak alkalinity adjustment, and derivatization reaction were performed according to step S2. High-performance liquid chromatography (HPLC) was then performed according to step S3 to calculate the actual recovered amount of topiramate in the sample. The sampling recovery rate was calculated as the ratio of the actual recovered mass to the theoretical spiked mass on the surface, with multiple parallel measurements taken as the average value. This experiment was used to evaluate the efficiency and stability of topiramate transfer from the equipment surface to the extraction system under single-wiping conditions.

[0056] Experimental Procedure for Aqueous Entrainment in Supernatant: The determination of the aqueous entrainment in the supernatant is used to evaluate the thoroughness of phase separation after salting-out. The specific experimental steps are as follows: After ultrasonic extraction as described in step S2.1 of the example, add the specified mass of the salting-out component in step S2.2, shake to mix, and then centrifuge. After centrifugation, allow to stand for 1 to 2 minutes to stabilize the separation interface. Carefully aspirate the organic phase supernatant using a graduated pipette or micro-pipette, and observe whether there is an aqueous tail or emulsion layer at the interface. Record the volume of visible aqueous entrainment in the supernatant, or indirectly calculate it by adding a small amount of anhydrous sodium sulfate to the supernatant and observing the amount of water extracted. Finally, convert the entrained aqueous volume into the aqueous entrainment amount corresponding to 20 mL of extract. Each experiment is repeated at least 3 times, and the average value is calculated. This index is used to evaluate the influence of the salting-out component and the synergistic component of ionic strength on the phase separation effect, and the instability caused by the presence of emulsion or aqueous entrainment in subsequent purification and quantification.

[0057] The experimental data are shown in Table 1.

[0058] Table 1: Examples 1 to 3 maintained a complete sampling, salting-out, and subsequent processing chain, thus maintaining a high recovery rate and low relative deviation in parallel measurements, indicating good repeatability and comparability under single sampling conditions. In Example 2, the addition of Tween-80 to the mixed extraction solvent further improved the recovery rate and reduced the relative deviation in parallel measurements. This indicates that Tween-80 enhances wetting and reduces interfacial energy, making it easier for topiramate to desorb from stainless steel or polymer surfaces and enter the extraction system, thereby improving the effective transfer of samples in a single sampling and reducing operational fluctuations. In Example 3, potassium chloride was introduced as a synergistic component for ionic strength after ultrasonic extraction. Following salting-out, the entrainment in the aqueous phase of the supernatant was significantly reduced, and the relative deviation in parallel measurements remained low. This indicates that ionic strength pre-adjustment and subsequent salting-out provide a continuous driving force, resulting in more thorough phase separation and inhibiting emulsion entrainment, thereby reducing the uncertainty of enriched extracts and improving repeatability. In Comparative Example 1, after removing the salting-out component, the amount of entrainment in the aqueous phase increased significantly, and the sampling recovery rate decreased. Simultaneously, the relative deviation of parallel measurements increased, demonstrating that salting-out stratification plays a necessary role in reducing entrainment, improving transfer efficiency, and controlling fluctuations. In Comparative Example 2, although the sampling recovery rate and the amount of entrainment in the aqueous phase remained unchanged, the relative deviation of parallel measurements increased significantly. This indicates that without using the unit area residual amount conversion and correction calculation, the results lack a unified scale and easily amplify operational differences, leading to decreased judgment stability.

[0059] Precision testing: Chromatographic conditions: A reversed-phase column (e.g., Phenomenex Luna® Omega 5µm PS C18 100Å, 4.6mm × 250mm) packed with octadecylsilane-bonded silica gel was used; the mobile phase was sodium acetate buffer (pH 4.7) and acetonitrile; the flow rate was 1.0 ml / min; the column temperature was 40℃; the detection wavelength was 360 nm; and the injection volume was 100 μl.

[0060] Gradient elution procedure: Solution preparation: 50% Acetonitrile: Measure 50 ml of chromatographic grade acetonitrile and mix thoroughly with 50 ml of water. This solution is used as the extraction solvent and diluent for the sample.

[0061] 0.5 mol / L sodium bicarbonate solution: Accurately weigh approximately 4.2 g of analytical grade sodium bicarbonate, dissolve it in water, and dilute to 100 mL. This solution provides the alkaline environment required for the derivatization reaction.

[0062] 2,4-Dinitrofluorobenzene solution: Accurately weigh approximately 0.1 g of chromatographically pure 2,4-dinitrofluorobenzene, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well. This solution is a derivatization reagent.

[0063] Phosphate buffer solution: Accurately weigh 1.61 g of disodium hydrogen phosphate and 0.48 g of potassium dihydrogen phosphate, dissolve in approximately 100 ml of water, and adjust the pH to approximately 7.0. This solution is used to terminate the derivatization reaction and provide a suitable injection environment.

[0064] Sodium acetate buffer solution (mobile phase A): Weigh 5.0 g of anhydrous sodium acetate, dissolve it in 1000 ml of water, and adjust the pH to 4.7 with glacial acetic acid.

[0065] Mobile phase B: chromatographic grade acetonitrile.

[0066] Reference solution: Weigh an appropriate amount of topiramate reference standard accurately, dissolve and dilute it with 50% acetonitrile to prepare a solution containing approximately 1.1 μg / ml of topiramate per ml.

[0067] Test solution: Accurately weigh approximately 11 mg of topiramate reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with 50% acetonitrile, and shake well. Accurately measure 10 ml of the solution and place it in a 25 ml volumetric flask. Dilute to the mark with 50% acetonitrile, and shake well. Take 0.5 ml of each of the above test solution and spread it evenly in 6 10 cm * 10 cm areas. After the solvent has evaporated, wipe the area with two pre-wetted cotton swabs (press them against the solvent bottle to remove excess solvent). Wipe each swab first in a horizontal serpentine pattern, then flip the swab and wipe in a vertical serpentine pattern. Then, place the two swabs into a stoppered test tube, accurately add 20 ml of 50% acetonitrile, seal, sonicate for 10 minutes, and filter through a 0.45 μm organic microporous membrane. Collect the filtrate to obtain the test solution.

[0068] Derivatization reaction procedure: Accurately measure 2 ml of blank solution, reference solution and each test solution into different 10 ml brown volumetric flasks. Accurately add 0.5 ml of 0.5 mol / L NaHCO3 solution and 3 ml of 2,4-dinitrofluorobenzene solution. Incubate in a 60℃ water bath for 30 min, then remove and cool. Dilute to the mark with phosphate buffer solution, shake well, and filter through a 0.45 μm organic microporous membrane. Collect the filtrate to obtain the final product.

[0069] The blank solution, reference solution, and test solution after the derivatization reaction were taken separately and injected for detection under the above chromatographic conditions. The results are shown in Table 2, and the chromatograms are shown in the figure. Figures 2 to 4 As shown.

[0070] Figure 2This is a chromatogram of a blank solution, representing the background response obtained under the same extraction, derivatization, and chromatographic conditions in a matrix system without topiramate and its derivatives. This chromatogram is mainly used to evaluate the specificity of the method and the system background interference. Chromatographically, no obvious characteristic peaks appeared near the expected retention time of the topiramate derivatives; only a small amount of baseline fluctuation or solvent front signal was observed. This indicates that the mobile phase system, derivatization reagent, and sample processing itself do not produce interfering peaks at the target retention time. The method has a good blank control basis, which is beneficial to the accuracy of subsequent quantitative calculations.

[0071] Figure 3 This is a chromatogram of the reference solution, representing the response of a reference solution obtained after derivatization of a known concentration of topiramate under the same chromatographic conditions. This chromatogram is used to establish the quantitative relationship between peak area and concentration, and to confirm the retention time and peak shape characteristics of the target derivative. The appearance of a single, symmetrical, and well-shaped characteristic peak at a specific retention time position indicates good chromatographic separation and that the system is suitable for quantitative analysis. This peak serves as a quantitative reference peak for accurate conversion of the residual topiramate concentration in the sample.

[0072] Figure 4 This is a chromatogram of the test solution, representing the detection result of the purified solution obtained after extraction, purification, derivatization, and volume adjustment following sampling from the equipment surface. This chromatogram is used to determine the presence of topiramate residue in the sample. The chromatogram shows a characteristic peak at the same retention time position as the reference standard, indicating the presence of topiramate derivative components in the sample. Furthermore, by comparing peak areas, the concentration of topiramate residue in the sample can be calculated. This concentration, combined with the sampling area and the adjusted volume, is then converted to the residue per unit area for determining cleanliness compliance. Therefore, this chromatogram demonstrates the applicability and quantitative capability of the method in actual samples.

[0073] Table 2 The above results demonstrate that the method provided by this invention has good precision.

[0074] Sensitivity test: Chromatographic conditions: Same as those for precision testing.

[0075] Solution preparation: Limit of quantitation solution: Take an appropriate amount of topiramate reference standard, accurately weigh it, dissolve it in 50% acetonitrile and dilute it quantitatively to a suitable concentration, inject it after derivatization reaction, record the chromatogram, and the limit of quantitation is when the signal-to-noise ratio is 10:1.

[0076] Limit of detection solution: Take an appropriate amount of topiramate reference standard, accurately weigh it, dissolve it in 50% acetonitrile and quantitatively dilute it to a suitable concentration, perform derivatization, inject the sample and determine the result, record the chromatogram, and the limit of detection is when the signal-to-noise ratio is 3:1.

[0077] The limit of quantitation (LOQ) and limit of detection (LOD) solutions after derivatization were taken and injected for detection under the above chromatographic conditions. The results are shown in Tables 3 and 4.

[0078] Table 3 Table 4 The results above show that both the limit of quantitation (LOQ) and the limit of detection (LOD) are lower than the chemical residue limit for topiramate (1.1 μg / ml), indicating that the method provided by this invention has high sensitivity.

[0079] 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, and improvements 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 topiramate cleaning chemical residues, characterized in that the steps include... include: S1. Take samples from a specified area to obtain samples, wherein the sampling steps include wiping sampling and / or rinsing sampling; S2. Add a mixed extraction solvent to the sample for extraction, filter the extract and adjust it to weak alkalinity, then add a derivatization reagent to react and obtain the purified solution to be tested. S3. Inject the purified solution to be tested into the chromatographic detection system for quantitative detection and output the detection results. The detection steps include chromatographic separation and signal response analysis.

2. The method for detecting topiramate cleaning chemical residues according to claim 1, characterized in that, Step S2 includes: S2.1 Place the sample in a stoppered centrifuge tube, use a pipette to precisely add the mixed extraction solvent for extraction, vortex for 30-60 seconds after adding, and then place it in an ultrasonic cleaner for ultrasonic extraction. The ultrasonic time is controlled to be 8-15 minutes. S2.2 After ultrasonic extraction, the extract is filtered through a 0.45-micron organic microporous membrane, and the filtrate is collected and used as the purified extract. S2.

3. Accurately measure the purified extract, add alkaline solvent to adjust the system to weak alkalinity, then add derivatization reagent solution and mix well, then place in a 60℃ constant temperature water bath to react in the dark for 25~35 min. S2.4 After the derivatization reaction is completed, remove the sample and cool it to room temperature. Then, dilute it to the mark with phosphate buffer solution and shake well to obtain the purified solution to be tested.

3. The method for detecting topiramate cleaning chemical residues according to claim 2, characterized in that, In step S2, the mixed extraction solvent includes at least one of acetonitrile-water mixed solvent, methanol-water mixed solvent, ethanol-water mixed solvent, isopropanol-water mixed solvent, and ethyl acetate-water mixed solvent; the alkaline solvent includes at least one of sodium bicarbonate solution, sodium carbonate solution, borate buffer, disodium hydrogen phosphate solution, and ammonia water; and the derivatizing reagent includes at least one of 2,4-dinitrofluorobenzene, p-nitrobenzenesulfonyl chloride, dansyl chloride, and 9-fluorenemethoxycarbonyl chloride.

4. The method for detecting topiramate cleaning chemical residues according to claim 1, characterized in that, Step S1 includes: S1.1 For flat, wipeable areas, use a dust-free cotton swab or polyester fiber wiping cloth for wiping and sampling. Before wiping, pre-wet the wiping material. The solvent used for pre-wetting should be the same as or compatible with the mixed extraction solvent. For areas that cannot be effectively wiped, use a rinsing sampling method. Use a fixed volume of rinsing solution to rinse repeatedly and recover the rinsing solution as the rinsing sample. The rinsing solution should be the same as or compatible with the mixed extraction solvent. S1.

2. A blank control sample shall be set up simultaneously in each sampling batch. The blank control sample shall include pre-wetting wiping material that has not been in contact with the equipment surface or rinsing liquid that has not been rinsed by the equipment.

5. The method for detecting topiramate cleaning chemical residues according to claim 1, characterized in that, Step S3 includes: S3.1 The purified solution to be tested is used for chromatographic detection. The chromatographic detection system is a high performance liquid chromatograph equipped with an ultraviolet detector. The chromatographic separation uses a reversed phase chromatographic column as the analytical column, and the column temperature is controlled at 35~45℃. S3.2 The mobile phase in the high performance liquid chromatograph includes an aqueous buffer and an organic solvent. An autosampler is used for injection, and the injection volume is controlled at 20~100μL. S3.

3. Prepare a reference solution of topiramate derivative. The reference solution is prepared according to the same derivatization process as the test purification solution, and the quantitative calculation is performed based on the correspondence between peak area and concentration to output the residual concentration of topiramate in the sample. S3.

4. The detected topiramate residual concentration is converted into residual amount per unit area based on the sampling area, and compared with the preset cleaning limit to output the cleaning qualification result.

6. The method for detecting topiramate cleaning chemical residues according to claim 5, characterized in that, In step S3, the aqueous buffer includes at least one of sodium acetate buffer, phosphate buffer, ammonium acetate buffer, ammonium formate buffer, and citrate buffer, and the organic solvent includes at least one of acetonitrile, methanol, ethanol, isopropanol, and tetrahydrofuran.

7. The method for detecting topiramate cleaning chemical residues according to claim 5, characterized in that, In step S3, the formula for calculating the residual amount per unit area is: In the formula, m represents the residual amount per unit area, with units of μg / cm². 2 ; The peak area is the peak area measured after derivatization of the test solution. The concentration of topiramate in the reference solution is expressed in μg / ml. The peak area is measured after derivatization of the reference solution; S represents the total volume of the sample after dilution during extraction, in ml; S represents the sampling area, in cm². 2 ; The measured residue per unit area is corrected using the sampling recovery rate, and the unit is μg / cm². 2 N represents the sampling recovery rate.

8. A method for detecting topiramate cleaning chemical residues according to any one of claims 5 or 7, characterized in that, In step S3, the preset cleanliness limit is 0.22 μg / cm³. 2 If the residual amount per unit area is less than or equal to the preset cleaning limit, the result of cleaning is qualified; if the residual amount per unit area is greater than the preset cleaning limit, the result of cleaning is unqualified.

9. A reagent kit, characterized in that, For implementing a method for detecting topiramate clean chemical residues as described in any one of claims 1 to 8, the kit contains a mixed extraction solvent and a derivatization reagent.

Images