In situ identification and quantification of genotoxic sulfonyl chloride impurity in topiramate (Topamax tablets) via LC-MS/MS

Abstract

This study reports a sensitive and selective method developed and validated for the detection of process-related genotoxic impurity (PGI) formed in situ during the synthesis of topiramate API. The method utilizes benzyl amine as a derivatizing agent to enhance sensitivity. of impurity. Chromatographic separation was performed on a Kromasil-C8 column (150 mm × 4.6 mm, 5 µm) with mobile phase A as 10 mM ammonium acetate containing 0.1 % formic acid in water and mobile phase B as acetonitrile (40:60 v/v). The flow rate was set at 1.2 mL/min and detection was carried out using an RI detector. Quantification was achieved using TQMS detection with electron spray ionization in MRM mode. The method exhibited excellent linearity in the range of 0.14–2.88 µg/mL with recovery between 96.82 % and 104.42 %. The method showed a detection limit of 0.0719 µg/mL and quantitation limit of 0.1438 µg/mL, making it suitable for trace-level analysis (< 1 ppm) of sulfonyl chloride in topiramate drug substance.

• •Derivatization with benzyl amine enhanced PGI detection sensitivity.
• •Separation using Kromasil-C8 column with acetonitrile/water buffer (40:60).
• •Detection via RI and quantification using TQMS in MRM mode.

Graphical abstract

A selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for detecting genotoxic sulfonyl chloride impurity in topiramate active pharmaceutical ingredient (API). The method involves in situ generation of the impurity followed by derivatization to enhance detection sensitivity and specificity. The validation process demonstrated the method's precision, accuracy, linearity, and robustness, ensuring its suitability for routine quality control and safety assessment of topiramate API.

Specifications table

Subject area:	Pharmaceutical Sciences
More specific subject area:	Analytical method development for genotoxic impurity detection in drug substances
Name of your method:	In situ identification and quantification of genotoxic sulfonyl chloride impurity in topiramate drug substance
Name and reference of original method:	No direct reference; method developed in-house as per ICH guidelines and optimized for topiramate drug substance
Resource availability:	All the resources needed to develop the procedure are provided in this article.

Background

Potential genotoxic impurities (PGIs) are harmful substances found in pharmaceutical products that have potential to cause genetic mutations, chromosomal damage, and cancer in humans [[1], [2]]. Restraining the formation of PGIs during the manufacturing process of APIs is challenging due to complex synthetic routes and use of multiple reactive reagents and intermediate. In accordance with regulatory requirements, these impurities must be controlled at the ppm level in the final products. Therefore, a major challenge for analytical chemists is to develop a suitable analytical method to quantify impurities and control their levels in APIs [[3], [4]]. According to the guidelines from the EMA [5], FDA) [6], ICH M7 [[7], [8]], and based on the threshold of toxicological concern (TTC), genotoxic impurities should be limited to 1.0 to 1.5 µg/day to ensure the safety and effectiveness of the final product [9].

Topiramate is an oral administered anticonvulsants or antiepileptic drugs used to treat epilepsy and migraines. During the manufacturing of topiramate, a genotoxic chloro analog, such as sulfonyl chloride impurity in topiramate, is formed at intermediate stages and appear at trace levels in the active pharmaceutical ingredient. This impurity is a suspected carcinogen classified as a potential genotoxic compound with a toxicological threshold limit < 1.5 µg/gm based on maximum daily intake of 1.0 g/day of topiramate [9]. Developing an analytical method capable of detecting genotoxic impurities at lower levels is a significant challenge. A literature survey revealed various HPLC, HPTLC, LC-MS, and ¹H NMR methods for analyzing topiramate in bulk drugs, formulations, and plasma [[10], [11], [12], [13], [14], [15], [16], [17], [18], [19]]. However, these methods primarily focus on topiramate quantification rather than regulatory control of PGIs. Currently, no suitable analytical method exists for detecting genotoxic chloro analog impurities at trace levels in topiramate. To bridge this gap, we adopted an LC-MRM approach to enhance the sensitivity of chloro analog impurities in topiramate. The developed method successfully detected sulfonyl chloride impurity in topiramate at low concentrations (0.07 µg/mL) with a shorter run time, offering a significant improvement in sensitivity for monitoring PGI limits in topiramate. Moreover, all crucial parameters related to the method performance, including limit of detection (LOD), limit of quantification (LOQ), specificity, recovery, reproducibility, and linearity have been established. The validated method serves as a reliable tool for routine quality control analysis and ensuring the safety and regulatory compliance of topiramate drug substances. The formation of sulfonyl chloride impurity during the manufacturing process of the topiramate API is illustrated in Fig. 1. Structure, carcinogenic potency category and AI value of potential genotoxic sulfonyl chloride impurity in topiramate is presented in Table 1.

Fig. 1.

The formation of sulfonyl chloride impurity during the manufacturing process of the topiramate API.

Table 1.

Structure, carcinogenic potency category and allowable intake value of potential genotoxic sulfonyl chloride impurity in topiramate.

Name of compounds	Structure	Molecular Formula	Molecular weight	Carcinogenic Potency category	Allowable intake (AI value)
Topiramate API		C12H21NO8S	339.36	–	1.0 g/day
Impurity 1 Topiramate - sulfonyl chloride		C12H19ClO8S	358.79	5	1.5 µg/gm.

Note: AI value predicted from Carcinogenic Potency Categorization for APIs at hypothetical risk of forming such PGIs.

Method details

Chemicals and reagents

Analytical-grade reagents with a purity of > 99.8 % were used. LC-MS grade methanol, acetonitrile, formic acid and ammonium acetate were procured from Thermo fisher (Mumbai. India). Milli-Q water collected from Milli-Q Plus water purification system (Millipore, Milford, MA, USA) was used throughout the analysis. The in-house preparation of topiramate-sulfonyl chloride in-situ impurity used as standard for the study.

Sample preparations

For the sample preparation, 1,4 dioxane is used as diluent.

• a)Preparation of blank solution: Add 0.25 mL of Benzyl amine into 10.0 mL volumetric flask containing about 2.0 mL of diluent. Stopper the flask and heat at 50 °C for 30.0 min into water bath. Cool and make up the volume up to the mark with diluent and mix well.
• b)Preparation of sulfonyl chloride impurity standard stock solutions: Weigh accurately 75.0 mg of the impurity and transfer it to a 50.0 mL volumetric flask. Add 20.0 mL of diluent, sonicate to dissolve the contents, and then dilute to volume with methanol to obtain a stock solution of 1500 µg/mL. From this, transfer 1.0 mL into a 50.0 mL volumetric flask and dilute with methanol to prepare a 30 µg/mL solution. Subsequently, take 1.0 mL of this solution and dilute to 100.0 mL with diluent to achieve a final concentration of 0.3 µg/mL.
• c)Preparation of sulfonyl chloride and topiramate sample solutions: To prepare the sulfonyl chloride sample solution (0.03 µg/mL), transfer 1.0 mL of the 0.3 µg/mL standard stock solution into a 10.0 mL volumetric flask containing 2.0 mL of diluent. Similarly, for the topiramate sample solution, accurately weigh about 200.0 mg of the topiramate sample and transfer it into a separate 10.0 mL volumetric flask containing 2.0 mL of diluent. In both flasks, 0.25 mL of benzylamine, stopper the flasks, and heat at 50 °C for 30 min in a water bath to complete derivatization process. After cooling, dilute each solution to volume with diluent and mix well.
• d)Preparation of Topiramate pharmaceutical dosage form sample solution (100µg/mL): Weighed four tablets (Topamac, 25 mg) of topiramate were taken into a cleaned mortar and grounded in powder. An amount of powder equivalent to 100 mg of topiramate was transferred into a 100 mL volumetric flask and added 70 mL of diluent into the flask. Sonicated the solution for 30 min and the volume was made up to the mark with diluent. The prepared solution was centrifuged at 4000 rpm for 5 min, and supernatant was filtered using a 0.22 µm membrane filter before injecting it into the LC-MS system.

Preparation of sulfonyl chloride impurity

β-d-fructopyranose (1) (5.5 g, 30.53 mmol) was dissolved in 25.0 mL of acetone in a 100 mL reaction flask. The flask was placed in an ice bath to maintain the temperature at 0 °C, and 1.0 mL of conc. H₂SO₄ was slowly added to the solution. The reaction mixture was stirred at 0 °C for 15.0 min, resulting in the formation of Bisacetonide (2). Subsequently, a mixture of 2.5 mL (30.87 mmol) of sulfuryl chloride and 2.5 mL of pyridine was prepared and slowly added drop wise to the reaction mixture. The reaction mixture was then stirred at room temperature for 90.0 min, resulting in the formation of sulfochloridate (3). The progress of the reaction was monitored using TLC.

Extraction and purification of sulfonyl chloride impurity

Upon completion of the reaction, the mixture was transferred into a 250.0 mL separatory funnel. To quench any unreacted sulfuryl chloride and remove excess pyridine, 25.0 mL of 10 % Na₂CO₃ solution was added to the reaction mixture. The mixture was then extracted by adding 25.0 mL of DCM to the separatory funnel and shake the mixture gently. Allow the layers to separate and collect the organic layer. Repeat the extraction step twice more using 25.0 mL of DCM each time to ensure complete extraction of the product. Then combine organic layers and transferred it into a 250 ml beaker. Add 5–10 g of anhydrous Na₂SO₄ and swirl the solution for 10–15 min to remove any residual moisture. The mixture was then filtered to obtain a clear filtrate. After that, organic layer was concentrated under reduce pressure at 50 °C using a vacuum evaporator to obtain the oily crude product. The crude material was then purified by flash chromatography using mixtures of cyclohexane : acetone (7:3), resulting in a pure compound 3.

Purity conformation & characterization of sulfonyl chloride impurity

After purification, the sample was analyzed by HPLC-RI to confirm its purity, which was found to be 99.1 %. Further structure elucidation was performed using mass and 1D-NMR (¹H, ¹³C) to provide the more support. Molecular formula : C₁₂H₁₉ClO₈S, Molecular weight: 358.79, Molecular ion peak [M + H]⁺ : 359.0, % HPLC-RI purity: 99.1 %. ¹H NMR (400 MHz, CDCl₃ ): δ (ppm): 4.599 - 4.573 (m, 1H, H₃), 4.490 - 4.365 (q, 2H, H₁₃), 4.278 - 4.209 (d, 1H, H₁), 4.187 - 3.876 (m, 1H, H₂), 3.848 - 3.714 (q, 2H, H₆), 1.504 - 1.297 (s, 12H, 4 CH₃). ¹³C NMR (100 MHz, CDCl₃ ): δ (ppm): 23.86 (C14, -CH₃), 24.90 (C15, -CH₃), 25.70 (C16, -CH₃), 26.33 (C17, -CH₃), 61.44 (C6, -CH₂), 66.94 (C1, -CH), 73.82 (C3, -CH), 76.819 (C2, -CH), 77.13 (C13, -CH₂), 99.93 (C4, 4° carbon), 109.14 (C8, 4° carbon), 109.54 (C11, −4° carbon). The representative chromatogram, mass spectrum, and ¹H & ¹³C NMR spectrum of the sulfonyl chloride impurity is also provided as supplementary material.

Instrumentations

AB Sciex QTRAP mass spectrometer (Model: API-4500, Foster City, USA), Agilent 1260 infinity HPLC system consisted of quaternary pump with inbuilt degasser (G1311B), auto sampler (G1329B), thermostat (G1330B ALS) and variable wavelength detector (G4212B) (Agilent Technologies, USA) were used for detection of the impurity. Data acquisition and processing were conducted using Analyst 1.7.3 software. pH of the buffer solution was tested with a Seven Excellence (Mettler, USA) digital pH meter. Deaeration was carried out using a USB 6.5 L ultrasonic bath (PCi Analyst). ¹H and ¹³C NMR were performed on a Bruker Avance Neo 400 MHz spectrometer (Bruker BioSpin Corp., USA). The instrument operated at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR utilizing DMSO‑d₆ solvents, with TMS being used as internal standard. The recorded chemical shifts for ¹H and ¹³C are reported on the δ scale in ppm, referenced to TMS (δ = 0.00) for ¹H NMR and DMSO‑d₆ (39.5 ppm) for ¹³C NMR.

Optimized conditions of LC-MS/MS

The chromatographic separation was performed using an Kromasil-C8 column (150 mm × 4.6 mm, 5 µm; GL Sciences, Japan). The mobile phase comprised two components: mobile phase A (10 mM ammonium acetate with 0.1 % formic acid) and mobile phase B (acetonitrile), initially mixed in a 40:60 (v/v) ratio. The flow rate was set to 1.2 mL/min, and the column temperature was maintained at 40 °C. A sample injection volume of 20 µL was used. Analysis was carried out using a isocratic program. A total run time for the chromatographic separation was 10 min. Optimized chromatographic parameters used for the separation of analytes are shown in Table 2. The MS/MS detection was carried out with an electron spray ionization (ESI) source with negative mode. The Q1 and Q3 values for Multiple Reaction Monitoring (MRM) were set at 428.3 and 186.0, respectively. The source parameters were as follows: Ion spray voltage, −4500 V, temperature, 550 °C, nebulizer gas (GS1), 50 psi; drying gas (GS2), 50 psi. During the analysis, impurity was identified based on their mass-to-charge (m/z) ratios, and the precursor and product ions were identified based on their mass abundance. The experiment was carried out in multiple reactions monitoring (MRM) mode, and it was used for scanning and acquiring mass data for quantification. The MRM parameters including quantifier ion, qualifier ion, declustering potential (DP), and collision energy (CE), were depicted in Table 3.

Table 2.

Optimized LC parameters of sulfonyl chloride impurity.

Chromatographic Parameter
Parameter	Value
Instruments	Agilent 1260 Infinity Quaternary pump (G1311B)
Sample Diluent	1,4 dioxane
Injection Volume	20 µL
Analytical Column	Kromasil-C8 column 150 mm x 4.6 mm, 5µ
Column Temperature	40 °C
Mobile Phase A	10 mM Ammonium acetate and 0.1 % formic acid in water
Mobile Phase B	Acetonitrile
Flow Rate	1.2 mL/min
Isocratic Programme	Time	A (%)	B (%)
	0	40	60
	10	40	60
Retention time of impurity	5.22

Table 3.

Mass spectrometer parameters of sulfonyl chloride impurity.

Parameters	Conditions
Instrument	AB Sciex QTRAP mass spectrometer
Ion source	Electrospray Ionization
Mode & Polarity	MRM - Negative
Curtain gas	30.0
CAD gas	Medium
Ion spray voltage	- 4500 V
Source temperature	550 °C
Gas 1	50.0
Gas 2	50.0
Dwell time	200 msec
Q1 and Q3 ion (m/z)	428.3 and 186.0 m/z
Decluster Potential (V)	−60.0
Entrance potential (V)	−9.0
Collision energy (V)	−40.0
Collision cell exit (V)	−13.0
Valco Programme	Time (minute)	Position of valve
	0–3.5	To waste
	3.5–10.0	To source

Acceptable criteria of method validation

The LC-MS/MS method was validated to assess system suitability and consistency. Key analytical parameters, including specificity, sensitivity (LOD and LOQ), linearity, recovery, and precision, were determined following ICH Q2(R1) guidelines [[20], [21], [22]]. A system suitability test was initially performed by injecting six replicate injections of a diluted standard solution of sulfonyl chloride impurity. The results were evaluated, and the % RSD was calculated, which should not exceed 15.0 %. Linearity was evaluated by preparing and injecting a series of standard solutions over a concentration range from LOQ to 200 % (0.003–0.058 µg/mL). Calibration curves were constructed using linear least-squares regression analysis, with the acceptance criterion of an R² value greater than 0.9999. Sensitivity was evaluated through LOD and LOQ calculations, based on signal-to-noise ratios of 3.0 and 10.0, respectively. Recovery was determined by spiking sulfonyl chloride at LOQ, 50 %, 100 %, 120 % and 200 % concentration levels. Method repeatability was evaluated by analyzing six replicates at 100 % concentration on the same day, expressed as % RSD. The benchtop stability of both the standard and test (derivatized) sulfonyl chloride impurity solutions was assessed at room temperature by analyzing samples at 0, 3, 6, 12, and 24 hrs. Benchtop stability was confirmed by a similarity factor of 0.98–1.02 and %RSD ≤ 2.0 % for the standard, and 90–110 % recovery with ±10 % difference for the test preparation over 24 hrs.

Method validation

Method design and refinement

The objective of this LC-MS/MS method was to achieve efficient separation of sulfonyl chloride impurity and topiramate peaks within a short runtime while maintaining system suitability parameters. At the initial stage, 50 µg/ml of sulfonyl chloride impurity standard was directly infused into the mass spectrometer to achieve maximum analyte response in both positive and negative ionization modes. In negative mode, the analyte peak was detected; however, its signal intensity decreased over time due to the analyte's instability in aqueous diluents such as water, methanol, and acetonitrile. To address this issue, derivatization trials were conducted by dissolving 1.0 mg of sulfonyl chloride impurity in 5.0 ml of 1,4-dioxane and adding 1.0 ml of benzyl amine as derivatizing agent. The reaction conditions were optimized by varying the concentration of derivatizing agent, temperature and reaction time. Different concentration of benzyl amine (0.20, 0.25, 0.30, 0.35 and 0.40 µg/mL) were tested, with the most intense peak observed at 0.30 µg/mL, which was selected as the optimum concentration. The effect of temperature on the derivatization process was evaluated at 30, 40, 50, and 60 °C, with 50 °C was identified as the optimum temperature. To further optimize the reaction time, different reaction time intervals (20, 30, 50 and 60 min) were tested, and a maximum yield was achieved at 30 min, which was chosen as optimum reaction time. Factors affecting reaction yield such as benzyl amine concentration, temperature ( °C), and reaction time (min), are illustrated in Fig. 2. Following the derivatization reaction, derivatized impurity was directly infused into the mass spectrometer to verify optimal analyte response in negative ionization mode. The MS/MS spectrum of sulfonyl impurity derivatized with benzyl amine is presented in Fig. 3. Once a strong response was obtained in the mass spectrometer, separation trials of the derivatized impurity and topiramate were conducted on the LC-MS system using isocratic mode. Based on the polarity of the derivatized impurity, a Symmetry C8 column (150 × 4.6 mm, 5 µm) and a Kromasil C8 column (150 × 4.6 mm, 5 µm) were tested to achieve optimal separation. Additionally, various buffer reagents such as 0.1 % trifluoroacetic acid and 10 mM ammonium acetate were evaluated. A mixture of 0.1 % formic acid, buffer and acetonitrile in isocratic program with Kromasil C8 column (150 × 4.6 mm, 5 µm) column was selected as optimal mobile phase and column that give the suitable selectivity and peak resolution in a shorter time. Final optimized chromatogram is shown in Fig. 4.

Fig. 2.

Optimization parameters affecting reaction yield: Benzylamine concentration, temperature, and time.

Fig. 3.

MS/MS spectraum of sulfonyl impurity derivatized with benzyl amine.

Fig. 4.

Final optimized chromatogram.

System suitability testing

System suitability testing was performed to ensure that the analytical system was appropriate for its intended application at the time of analysis. This was evaluated by analyzing six replicate injections (n = 6) of a test solution containing sulfonyl chloride impurity. The retention time and % RSD of the peak areas for sulfonyl chloride impurity were found to be 5.33 min and 0.94 %, respectively. As the % RSD was within acceptable limits, the results confirmed the suitability of the system for analysis. A summary of the results is provided in Table 1.

Specificity

The specificity of the method was evaluated by assessing potential interfering components in the LC-MS/MS spectrogram. A 20 µL solution of blank, test standard solutions of sulfonyl chloride impurity and topiramate, and a spiked sample (containing both topiramate and sulfonyl chloride impurity) were injected separately into the LC-MS/MS system. The results demonstrated no interference from any interfering components, and all analyte peaks remained distinct and unaltered. The spectrogram obtained are shown in Fig. 5. The results revealed that there was no interference of the topiramate with sulfonyl impurity peaks and hence the specificity of the developed method was proven.

Fig. 5.

Representative chromatograms of (a) blank, (b) test sample of in-situ impurity, (c) test sample of topiramate (d) Spiked sample (topiramate test sample + impurity test sample).

Sensitivity

The Limit of Detection (LOD) and Limit of Quantification (LOQ) values for the sulfonyl chloride impurity were determined based on the analyte response. LOD and LOQ values of sulfonyl chloride impurity with respect to topiramate were found to be 0.075 and 0.15 µg/mL, respectively. The LOQ precision was assessed by conducting six replicate injections of LOQ samples and analyzing their results. The % RSD for these six measurements of LOQ precision was 0.85. The obtained LOD and LOQ values, along with their signal-to-noise ratios, are presented in Table 4. A typical chromatogram for LOD and LOQ are shown in Fig. 6.

Table 4.

LC- MS/MS method validation parameters for sulfonyl chloride impurity.

Parameter	Typical acceptance criteria	Result
System suitability	RSD (%) for peak area response (n = 6) should be not >15.0 %	0.94 %
Specificity	Interference from blank and sample matrix	No interference
LOD	S/N ration should be not <3	0.075 µg/mL (Respect to drug)
LOQ	S/N ration should be not <10	0.15 µg/mL (Respect to drug)
	RSD for six replicate (n = 6) injections of LOQ solution should be ≤ 15.0 %	0.85 %
Linearity	Range (LOQ to 200 %)	0.003–0.058 µg/mL
	coefficient of determination (R2) > 0.990	0.9999
Accuracy	% recovery for five different sample injections of LOQ to 200 % solutions (n = 3) should fall within the range of 70 % to 130 %, with an RSD <15 %.	70 - 130 % 0.41 to 2.53 %
Precision	RSD (%) for six preparations at 100 % level should be ≤ 15.0 %	0.64 %

Note: R² = determination of coefficients, LOD = Limit of Detection, LOQ = Limit of quantification, RSD = Relative standard deviation,.

Fig. 6.

A typical chromatograms of (a) Limit of Detection, (b) Limit of Quantification.

Linearity

The linearity of the sulfonyl chloride impurity was satisfactorily demonstrated with a seven-point calibration graph, ranging from the LOQ to 200 % of the specification level (actual concentration : 0.003, 0.007, 0.014, 0.023, 0.029, 0.035, 0.043, and 0.06 µg/mL). The slope, intercept and correlation coefficient values were derived from linear least-square regression analysis and the data is presented in Table 4. It reveals that an excellent correlation existed between the peak areas concentration of sulfonyl chloride impurity.

Precision

A precision study was conducted by spiking test samples (n = 6) of topiramate with a known amount of sulfonyl chloride impurity (0.03 µg/mL) to evaluate method precision. The % RSD obtained for method precision was 0.64 %. These results demonstrate that the method is sufficiently precise, with detailed precision data provided in Table 4.

Accuracy

The recovery testing involved spiking a known quantity of sulfonyl chloride impurity with topiramate API at various levels (LOQ = 0.14 µg/mL, 50 % = 0.72 µg/mL, 100 % = 1.44 µg/mL, 120 % = 1.73 µg/mL, and 200 % = 2.88 µg/mL of the specification with respect to topiramate). Each concentration level was injected triplicate into the LC-MS. The acceptance criteria for recovery was established at 70 - 130 % with RSD < 15 %. The % recovery values obtained for the sulfonyl chloride impurity ranged from 97.64 % to 102.76 %, which are within the acceptance criteria. The % RSD values of recoveries for the sulfonyl chloride impurity ranged from 0.41 % to 2.53 % at LOQ to 200 % levels. The % recovery results are shown in Table 4.

Benchtop stability of standard and test preparations

The benchtop stability of both standard and test (derivatized) sulfonyl chloride impurity solutions was evaluated by preparing as per above described method and stored at ambient temperature. Samples were analyzed at 0 and 24 hrs to monitor any changes in response. Stability of standard solution was assessed using six replicate injections (n = 6) at 0 and 24 hrs, based on mean peak area, % RSD, and similarity factor. The standard solution showed minimal change in peak area with a similarity factor of 0.99 and low % RSD values (≤ 3.26), indicating consistent analytical performance. The test preparation was assessed through % recovery and % difference from initial response. The derivatized impurity showed acceptable recovery of 101.55 % and an % difference of 4.05 %, within the predefined acceptance criteria. These results confirm that both the standard and test preparations are stable for up to 24 hrs on the benchtop (Table 5xsy) [[23], [24], [25]].

Table 5.

Summary of solution stability data for standard and derivatized sulfonyl chloride impurity.

Component	Intervals	Recovery %	% Difference	Average Peak Area	% RSD	Similarity Factor
Sulfonyl chloride impurity	Initial	–	–	191,519	3.26	1.00
	24	–	–	189,688	2.94	0.99
Derivatized Sulfonyl chloride impurity	Initial	97.59	0	–	–	–
	24 h	101.55	4.05	–	–	–

Application of the method for real sample analysis

Topiramate in bulk-scale batches (3 batches) and pharmaceutical dosage forms have been obtained, and their test sample solutions have been prepared as described. The sample solutions were subjected to the developed and validated LC–MS/MS method to identify the sulfonyl chloride impurity. The results indicated that the sulfonyl chloride impurity was not present in the examined samples. To enhance understanding, sulfonyl chloride impurity were added to the test samples. The analysis showed that these sulfonyl chloride impurity was well separated and did not interfere with sample matrix. As a result, the method proved to be reliable and accurate for determining sulfonyl chloride impurity in bulk and formulated samples.

Comparison of the present method with the earlier reported methods

The analytical performance of the proposed LC-MS/MS method was evaluated and compared with previously reported techniques in terms of sensitivity, run time, and selectivity (Table 6). Unlike earlier methods, which focused developed on quantification of topiramate in different matrices, the present method is specifically developed for the identification and quantification of a genotoxic sulfonyl chloride impurity in topiramate drug substance, in accordance with ICH M7 guidelines and fully validated as per ICH Q2(R2). Moreover, none of the existing reported methods address this specific impurity. The proposed method offers shorter run time (10 min) and uses low environmental impact reagents, and demonstrates improved selectivity, high accuracy, and cost-effectiveness, making it valuable advancement for genotoxic impurity control in pharmaceutical.

Table 6.

Performance comparison of proposed method with the previously developed analytical techniques.

Author	Analytical Method	Sensitivity	Run Time (min)	Selectivity	Remarks	Ref.
Proposed method	LC-MS/MS (MRM) with benzylamine derivatization	LOD ∼ 2.94 (µg/mL) LOQ ∼ 11.15 (µg/mL)	∼ 20	∼ Selective for sulfonyl chloride impurity in API	Designed for PGI detection under ICH M7	–
Pinto et al. (2018)	HPLC with Mixed-mode column (MMC) and CAD detection	LOD ∼ 2.94 (µg/mL) LOQ ∼ 11.15 (µg/mL)	∼ 20	∼ Selective for DPs of topiramte	Targeted DPs, not PGIs	[10]
Martinc et al. (2014)	HPLC-Fluorescence with NBD-Cl derivatization	LLOQ ∼ 0.5 (µg/mL)	∼ 12.2	∼ Selective for topiramate quantification	Designed for therapeutic drug monitoring	[11]
Majnooni et al. (2014)	HPLC-UV after NBD-Cl pre-column derivatization	LOD ∼ 0.5 (µg/mL) LOQ ∼ 1.0 (µg/mL)	∼ 3.0	∼ Selective for topiramte in formulations	Avoids use of RID, optimized for dissolution testing	[12]
Munshi et al. (2018)	HPTLC-Densitometric with Derivatization (dip method)	LOD ∼ 0.36 (µg/band) LOQ ∼ 1.1 (µg/band)	–	∼ Selective for DPs of topiramte	Cost-effective method for therapeutic drug monitoring in biological samples	[13]
Sangamithra et al. (2020)	LC-MS/MS	LOD ∼ 0.5 (ng/mL) LOQ ∼ 1.0 (ng/mL)	∼ 2.0	∼ Selective for topiramate quantification	Ultra-sensitive, rapid method for topiramte assay in formulations	[14]
Milosheska et al. (2017)	LC-MS/MS (MRM, negative mode) for topiramate + metabolites	LOQ ∼ 0.10 (µg/mL)	∼ 5.5	∼ Selective for plasma validation plasma, no matrix effect	Fully validated with stable-isotope internal standard	[15]
Matar (2010)	LC-MS/MS (MRM with ESI) for TPM in plasma using TPM-d₁₂ as IS	ULOQ ∼ 0.5 (µg/mL)	∼ 5.0	∼ Selective for plasma validation plasma, no matrix effect	Validated for clinical topiramate	[16]
Contin et al. (2001)	HPLC-Turbo Ion Spray MS (positive ion mode)	LLQ ∼ 0.25 (µg/mL)	∼ 2.1	∼ Selective for plasma validation	Simple and fast method with high recovery (92–95 %)	[17]
Ni Yang et al. (2015)	LC-MS/MS with ESI positive/negative ion-switching MRM	LOQ ∼ 1.0 (µg/mL)	∼ 1.87	∼ Selective for plasma validation	Pharmacokinetic application; two-drug monitoring in plasma	[18]
Qin et al. (2019)	Quantitative ¹H—NMR spectroscopy	LOD ∼ 0.04 (mg/mL) LOQ ∼ 0.16 (mg/mL)	–	∼ Selective for API quantification	Accurate for API assay	[19]

Disclaimer

In this study, we have developed a simple LC-MS/MS approach that is capable of quantifying potential genotoxic sulfonyl chloride impurity (PGI) formed in situ during the synthesis of topiramate API using the negative ionization mode with multiple reaction monitoring (MRM) at TTC level. The method is validated as per ICH recommendations and it is found to be specific and linear over the specified concentration range. Method is highly sensitive with a limit of detection (LOD) of 0.075 µg/mL. The method is fully validated and presents good linearity, specificity, accuracy, and precision. Besides, the method presented here could be very useful for the determination of potential genotoxic sulfonyl chloride impurity during its manufacture and also feasible for routine quality control testing.

Ethics statement

This study did not involve any human and animal subjects.

Finding

This research was self-funded by the authors. No external financial support was received for the conduct of this study.

CRediT authorship contribution statement

Hitesh Thumbar: Project administration, Methodology, Investigation, Formal analysis. Jayesh Dhalani: Formal analysis, Data curation. Hetal Patel: Investigation, Methodology, Funding acquisition. Bhavin Dhaduk: Supervision, Project administration, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.