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<p>Nitrosamine drug substance-related impurities (NDSRIs) have emerged as a critical regulatory concern due to their potential genotoxicity at trace levels, necessitating highly sensitive and scientifically robust analytical methodologies. Despite the widespread use of levofloxacin (LOX), no validated analytical method has been reported for the simultaneous determination of LOX-specific NDSRIs. Although several LC–MS/MS methods have recently been reported for NDSRIs associated with other pharmaceutical compounds, these studies primarily focused on compound-specific impurity monitoring and did not address levofloxacin-derived NDSRIs. Furthermore, most reported methodologies emphasize analytical sensitivity without integrating toxicological risk assessment and sustainability evaluation. In this study, a highly sensitive and selective LC–TQ–MS/MS method was developed and validated for the simultaneous trace-level quantification of N-nitroso methyl piperazine (N-NMP) and N-nitroso desmethyl levofloxacin (N-NDLOX) in LOX drug substance and tablet formulations. A systematic risk-based analytical development strategy was employed to define the Analytical Target Profile (ATP) and guide method development and optimization. Structural confirmation of the synthesized NDSRIs was achieved using advanced spectroscopic techniques, including <sup>1</sup>H–<sup>15</sup>N HMBC NMR. The developed method demonstrated excellent sensitivity, with limits of quantification of 0.6&#xa0;ng&#xa0;mL⁻<sup>1</sup> for N-NMP and 1.0&#xa0;ng&#xa0;mL⁻<sup>1</sup> for N-NDLOX, enabling reliable detection well below the proposed interim control limits. The method exhibited strong linearity (R<sup>2</sup> &gt; 0.998), high accuracy (93–100%), and robust precision (CV &lt; 10%), confirming its suitability for routine quality control applications. To support regulatory risk assessment, complementary (Q)-SAR analysis and carcinogenic potency categorization (CPCA) were integrated to establish scientifically justified interim control limits for LOX-associated NDSRIs. The sustainability of the developed method was further evaluated using a multi-metric framework incorporating AGREE, GAPI, RAPI, and the Environmental, Performance, and Practicality Index (EPPI). The results demonstrated a balanced analytical profile, with moderate environmental impact (AGREE score: 0.62; EI: 75.8%) and high practical applicability (PPI: 87.0%), resulting in an overall EPPI score of 81.4%. This study presents a unified analytical–toxicological–sustainability framework for NDSRI assessment, representing a shift from conventional method development toward performance-driven sustainable analytical design. The proposed method is suitable for regulatory implementation and provides a scalable strategy for controlling structurally complex nitrosamine impurities in pharmaceutical systems.</p>

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Trace-level quantification of levofloxacin NDSRIs using LC–TQ–MS/MS: a multi-metric sustainability and integrated toxicological–analytical framework

  • 期刊论文

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  • 发表日期：

    2026年7月10日

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  • Srinivas Nakka,
  • Vishnu Murthy Marisetti,
  • Surendra Babu Manabolu Surya,
  • Hemanth Kumar Chanduluru

摘要

Nitrosamine drug substance-related impurities (NDSRIs) have emerged as a critical regulatory concern due to their potential genotoxicity at trace levels, necessitating highly sensitive and scientifically robust analytical methodologies. Despite the widespread use of levofloxacin (LOX), no validated analytical method has been reported for the simultaneous determination of LOX-specific NDSRIs. Although several LC–MS/MS methods have recently been reported for NDSRIs associated with other pharmaceutical compounds, these studies primarily focused on compound-specific impurity monitoring and did not address levofloxacin-derived NDSRIs. Furthermore, most reported methodologies emphasize analytical sensitivity without integrating toxicological risk assessment and sustainability evaluation. In this study, a highly sensitive and selective LC–TQ–MS/MS method was developed and validated for the simultaneous trace-level quantification of N-nitroso methyl piperazine (N-NMP) and N-nitroso desmethyl levofloxacin (N-NDLOX) in LOX drug substance and tablet formulations. A systematic risk-based analytical development strategy was employed to define the Analytical Target Profile (ATP) and guide method development and optimization. Structural confirmation of the synthesized NDSRIs was achieved using advanced spectroscopic techniques, including 1H–15N HMBC NMR. The developed method demonstrated excellent sensitivity, with limits of quantification of 0.6 ng mL⁻1 for N-NMP and 1.0 ng mL⁻1 for N-NDLOX, enabling reliable detection well below the proposed interim control limits. The method exhibited strong linearity (R2 > 0.998), high accuracy (93–100%), and robust precision (CV < 10%), confirming its suitability for routine quality control applications. To support regulatory risk assessment, complementary (Q)-SAR analysis and carcinogenic potency categorization (CPCA) were integrated to establish scientifically justified interim control limits for LOX-associated NDSRIs. The sustainability of the developed method was further evaluated using a multi-metric framework incorporating AGREE, GAPI, RAPI, and the Environmental, Performance, and Practicality Index (EPPI). The results demonstrated a balanced analytical profile, with moderate environmental impact (AGREE score: 0.62; EI: 75.8%) and high practical applicability (PPI: 87.0%), resulting in an overall EPPI score of 81.4%. This study presents a unified analytical–toxicological–sustainability framework for NDSRI assessment, representing a shift from conventional method development toward performance-driven sustainable analytical design. The proposed method is suitable for regulatory implementation and provides a scalable strategy for controlling structurally complex nitrosamine impurities in pharmaceutical systems.