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<p>The present study describes the development of a green, stability-indicating HPLC method coupled with LC–MS/MS characterization for degradation products (DPs) of darolutamide. The chromatographic separation was achieved on a phenyl-hexyl column (250 × 4.6&#xa0;mm, 5&#xa0;µm) using an isocratic mobile phase of ethanol and 5&#xa0;mM ammonium formate with 0.1% formic acid (55:45, v/v, pH 3.6) at 0.8&#xa0;mL/min, with UV detection at 238&#xa0;nm. The method provides sharp, symmetrical peaks with retention times of 5.8&#xa0;min, 4.2&#xa0;min, 1.59&#xa0;min, and 2.96&#xa0;min for darolutamide, nitroso impurity, and nitroso impurities 1 and 2. The forced degradation studies reveal that darolutamide was most susceptible to acid, base, and oxidative stress. These stress degradation lead to the formation of six novel DPs (DP1-DP6). The structural elucidation by LC–MS/MS confirms that DP1 and DP2 were nitrile hydrolysis products (amide and carboxylic acid derivatives). The DP3 was arising from alcohol oxidation to ketone, whereas DP6 results from acid-catalyzed dehydration to an alkene. The DP4 and DP5 emerge from sequential amide bond cleavage and side-chain loss from the darolutamide. The in silico toxicity prediction classifies all DPs in toxicity classes 4–5 (LD₅₀ = 1000–2200&#xa0;mg/kg), with low acute toxicity. The method validation confirms the excellent linearity (r<sup>2</sup> &gt; 0.996), high precision (%RSD &lt; 1.0%), accuracy (recoveries 99.4–101.1%), robustness, and sensitivity (LOD as low as 0.015&#xa0;µg/mL). The method’s greenness and sustainability were proved with an AGREE score of 0.83, ComplexGAPI, and an RGB whiteness score of 80.1%. This study highlights the compliance of the method with green and white analytical chemistry. This work uniquely addresses the nitroso impurities of regulatory concern with superior environmental and analytical performance. In conclusion, the proposed method was a sustainable, reliable, and regulatory-compliant approach for routine impurity profiling and stability assessment of darolutamide.</p>

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Analytical Green and White HPLC Method for the Quantification of Nitroso Impurities in Darolutamide with MS/MS Characterization and In silico Toxicity Assessment of Degradation Products

  • 期刊论文

  • 发表日期：

    2026年2月8日

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  • Katari Ravi Babu,
  • Gowri Sankar Reddipalli

摘要

The present study describes the development of a green, stability-indicating HPLC method coupled with LC–MS/MS characterization for degradation products (DPs) of darolutamide. The chromatographic separation was achieved on a phenyl-hexyl column (250 × 4.6 mm, 5 µm) using an isocratic mobile phase of ethanol and 5 mM ammonium formate with 0.1% formic acid (55:45, v/v, pH 3.6) at 0.8 mL/min, with UV detection at 238 nm. The method provides sharp, symmetrical peaks with retention times of 5.8 min, 4.2 min, 1.59 min, and 2.96 min for darolutamide, nitroso impurity, and nitroso impurities 1 and 2. The forced degradation studies reveal that darolutamide was most susceptible to acid, base, and oxidative stress. These stress degradation lead to the formation of six novel DPs (DP1-DP6). The structural elucidation by LC–MS/MS confirms that DP1 and DP2 were nitrile hydrolysis products (amide and carboxylic acid derivatives). The DP3 was arising from alcohol oxidation to ketone, whereas DP6 results from acid-catalyzed dehydration to an alkene. The DP4 and DP5 emerge from sequential amide bond cleavage and side-chain loss from the darolutamide. The in silico toxicity prediction classifies all DPs in toxicity classes 4–5 (LD₅₀ = 1000–2200 mg/kg), with low acute toxicity. The method validation confirms the excellent linearity (r2 > 0.996), high precision (%RSD < 1.0%), accuracy (recoveries 99.4–101.1%), robustness, and sensitivity (LOD as low as 0.015 µg/mL). The method’s greenness and sustainability were proved with an AGREE score of 0.83, ComplexGAPI, and an RGB whiteness score of 80.1%. This study highlights the compliance of the method with green and white analytical chemistry. This work uniquely addresses the nitroso impurities of regulatory concern with superior environmental and analytical performance. In conclusion, the proposed method was a sustainable, reliable, and regulatory-compliant approach for routine impurity profiling and stability assessment of darolutamide.