TRACE LEVEL QUANTIFICATION OF C1-C3 ALKYL CHLORIDE GENOTOXIC IMPURITIES IN TRIMETAZIDINE DI HYDROCHLORIDE DRUG SUBSTANCE USING STATIC HEADSPACE GAS CHROMATOGRAPHY

RAJAVENKATA PRASAD PATHA; KARUNAKAR DASA; RAMA DEVI BHOOMIREDDY; RAVI KIRAN PANCHAKARLA; RAJ KUMAR KOKKONDA

doi:10.22159/ajpcr.2025v18i3.53750

Authors

RAJAVENKATA PRASAD PATHA Department of Chemistry, Jawaharlal Nehru Technological University, Hyderabad, Telangana, India. Analytical Research and Development, Sai Life Sciences Limited, Genome Valley, Hyderabad, Telangana, India https://orcid.org/0009-0007-5330-8411
KARUNAKAR DASA Department of Chemistry, Government Degree College, Kukatpally, Telangana, India.
RAMA DEVI BHOOMIREDDY Department of Chemistry, Jawaharlal Nehru Technological University, Hyderabad, Telangana, India
RAVI KIRAN PANCHAKARLA Analytical Research and Development, Sai Life Sciences Limited, Genome Valley, Hyderabad, Telangana, India https://orcid.org/0000-0003-1074-0937
RAJ KUMAR KOKKONDA Analytical Research and Development, Sai Life Sciences Limited, Genome Valley, Hyderabad, Telangana, India

DOI:

https://doi.org/10.22159/ajpcr.2025v18i3.53750

Keywords:

Genotoxic impurities,, Gas chromatography, Method validation, Chloroalkanes, Drug substance

Abstract

Objectives: The objective of this study was to develop and validate a gas chromatography-headspace (GC-HS) method for the quantification of genotoxic alkyl chloride impurities (chloromethane, ethyl chloride, and isopropyl chloride) in trimetazidine dihydrochloride, ensuring compliance with International Council for Harmonization M7 guidelines.

Methods: A GC-HS method was optimized using a DB-1 column (60 m×0.32 mm, 3.0 μm) with nitrogen as the carrier gas. Key parameters included HS conditions with an oven temperature of 95°C, sample line temperature of 105°C, and transfer line temperature of 115°C, along with a split ratio of 1:10 and a flow rate of 10.2 psi. The oven temperature program was set to start at 40°C for 15 min, followed by an increase of 30°C/min to 250°C, held for 15 min. Method validation assessed linearity, detection limits, quantification limits, accuracy, precision, and solution stability.

Results: The method exhibited excellent linearity (r²>0.999), low limits of detection (0.6 ppm) and quantification (1.8 ppm), and high accuracy (91.0–114.0% recovery). Precision was confirmed with relative standard deviations below 5%. Sample solutions remained stable for up to 48 h, demonstrating the method’s robustness and reliability for routine analysis.

Conclusion: The developed GC-HS method is a robust, accurate, and regulatory-compliant approach for the trace-level quantification of genotoxic alkyl chloride impurities in trimetazidine dihydrochloride, ensuring the safety and quality of the pharmaceutical product.

Downloads

Download data is not yet available.

References

Yang J, Zhang L, Liu C, Zhang J, Yu S, Yu J, et al. Trimetazidine attenuates high-altitude fatigue and cardiorespiratory fitness impairment: A randomized double-blinded placebo-controlled clinical trial. Biomed Pharmacother. 2019;116:109003. doi: 10.1016/j. biopha.2019.109003, PMID 31125823

McClellan KJ, Plosker GL. Trimetazidine. A review of its use in stable angina pectoris and other coronary conditions. Drugs. 1999;58(1):143-57. doi: 10.2165/00003495-199958010-00016, PMID 10439934

Vitale C, Marazzi G, Pelliccia F, Volterrani M, Cerquetani E, Spoletini I,

et al. Trimetazidine improves exercise performance in patients with peripheral arterial disease. Pharmacol Res. 2011;63(4):278-83. doi: 10.1016/j.phrs.2011.01.003, PMID 21220024

Sigmund G, Koch A, Orlovius AK, Guddat S, Thomas A, Schänzer W, et al. Doping control analysis of trimetazidine and characterization of major metabolites using mass spectrometric approaches. Drug Test Anal. 2014;6(11-12):1197-205. doi: 10.1002/dta.1680, PMID 24913825

Giordani A, Kobel W, Gally HU. Overall impact of the regulatory requirements for genotoxic impurities on the drug development process. Eur J Pharm Sci. 2011;43(1-2):1-15. doi: 10.1016/j.ejps.2011.03.004, PMID 21420491

Yang Q, Haney BP, Vaux A, Riley DA, Heidrich L, He P, et al. Controlling the genotoxins ethyl chloride and methyl chloride formed during the preparation of amine hydrochloride salts from solution and ethanol and methanol. Org Process Res Dev. 2009;13(4):786-91. doi: 10.1021/op9000737

Müller L, Mauthe RJ, Riley CM, Andino MM, Antonis DD, Beels C, et al. A rationale for determining, testing, and controlling specific impurities in pharmaceuticals that possess potential for genotoxicity. Regul Toxicol Pharmacol. 2006;44(3):198-211. doi: 10.1016/j. yrtph.2005.12.001, PMID 16412543

Snodin DJ. Genotoxic impurities: From structural alerts to qualification. Org Process Res Dev. 2010;14(4):960-76. doi: 10.1021/op100118e

European Medicines Agency (EMEA), Committee for Medicinal Products for Human Use (CHMP). Guideline on the Limits of Genotoxic Impurities, CPMP/SWP/5199/02. In: Guideline on Genotox Impurities-Final-SWP Number-Superseded Watermarked Jul 2019 doc; 2006. Available from: https://www.europa.eu

USFDA. S2 (R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. ICH S2 (R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use-Scientific Guideline. In: European Medicines Agency (EMA); 2012. Available from: https://www.europa.eu

ICH. M7 (R1): Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk; 2017. Available from: https://database.ich.org/sites/default/files/m7_ r1_guideline.pdf

Sultana S, Nagarajan B. Simultaneous trace-level determination of benzene and 1,2-dichloroethane by GC-HS/GC-MS in several pharmaceutical drug substances. Int J Appl Pharm. 2019;11(1):82-8. doi: 10.22159/ijap.2019v11i1.28983

Aparna K, Rachel KV, Rao KM. Quantitative determination of methyl-4-chlorobutyrate, a potential genotoxic impurity, content in moxifloxacin HCl by GC-EI-MS. Int J Appl Pharm. 2024;16(5):234-41. doi: 10.22159/ijap.2024v16i5.51551

Hughes RA, Knighton WB, Grimsrud EP. Enhancement of electron-capture detection of methyl bromide in air by iodination. J Chromatogr A. 1999;852(2):535-43. doi: 10.1016/S0021-9673(99)00649-4, PMID 10481990

Narapereddy KP, Alladi DS. Development and validation of determination of genotoxic impurity bromoethane in vigabatrin drug substance using head space gas chromatographic method [HS-GC]. Pharmacia. 2023;70(1):203-07. doi: 10.3897/pharmacia.70.e97339

Van Wijk AM, Beerman B, Niederländer HA, Siebum AH, De Jong GJ. A new approach for generic screening and quantitation of potential genotoxic alkylation compounds by pre-column derivatization and LC-MS/MS analysis. Anal Bioanal Chem. 2011;400(5):1375-85. doi: 10.1007/s00216-011-4901-y, PMID 21445660

Rajesh Varma B, Srinivas Rao B, Kapavarapu MN, Varaprasad Reddy M. Assessment of gas chromatography methodology approach for the trace evaluation of carcinogenic impurity, methyl chloride, in trimetazidine dihydrochloride. Ann Pharm Fr. 2023;18(1):64-73. doi: 10.1016/j. pharma.2022.06.012

ICH Q2A(R1). Validation of analytical Procedures: Text and Methodology; 2005. Available from: https://database.ich.org/sites/ default/files/q2%28r1%29%20guideline.pdf

Patha RP, Dasa K, Bhoomireddy RD, Thumu SR. In-silico toxicity assessment and trace level quantification of veratryl chloride, a potential genotoxic impurity in ivabradine hydrochloride using LC-MS/MS. Int J Pharm Sci Drug Res. 2023;15(4):488-93. doi: 10.25004/ IJPSDR.2023.150413

US FDA. Analytical Procedures and Methods Validation for Drugs and Biologics; 2015. Available from: https://www.fda.gov/files/drugs/ published/analytical-procedures-and-methods-validation-for-drugs-and-biologics.pdf