A sustainable and novel LC-TQ-MS/MS method for quantifying mutagenic Ketoconazole-NDSRIs aligned with green and white analytical chemistry principles

Abstract

Fungal infections affect millions of people globally, ranging from minor skin conditions to life-threatening diseases. Ketoconazole (KCZ)MS, a potent synthetic imidazole, remains widely prescribed. This study reports the synthesis of N-nitrosoacetylpiperazine (N-NAP), a nitroso derivative of a KCZ-degradant, with the N-nitroso group confirmed by ¹H–¹⁵N HMBC spectroscopy, and their genotoxic potential was evaluated using two (Q)SAR tools. The identified KCZ NDSRIs are categorized as class-3 as per QSAR, and the predictions showed as genotoxic positive (Cohort-of-concern). The acceptable intake (AI) levels of both nitrosamines were determined using the carcinogenic potency categorization approach (CPCA) and belonged to potency category-3. Following ICH Q14 recommendations, the LC-TQ-MS/MS method was optimized for the detection and quantification of KCZ-NDSRIs at trace amounts in KCZ pharmaceuticals. This study advances two Sustainable Development Goals (SDGs). It supports SDG 3 (good health and well-being, targets 3.8 & 3.d) by utilizing (Q)-SAR and CPCA analyses and contributes to SDG 9 (industry, innovation, and infrastructure, target 9.4) via a validated LC-TQ-MS/MS method for quantifying NDSRIs as per ICH Q2(R2). The chromatographic separation was optimized using a Waters X-bridge BEH C18 column (150 mm × 2.1 mm, 2.5 µm) with gradient elution. Mobile phases consisted of 0.1% formic acid in water and acetonitrile with a column oven temperature of 40 °C and a pump flow rate of 0.4 mL min⁻¹. Aligned with ICH Q14, this approach defines the analytical target profile (ATP) and established conditions (ECs) and incorporates risk assessment and reporting categories. The robustness and validation of the current method were executed following the ICH Q2(R2) guidelines. The sustainability of the proposed method was demonstrated using cutting-edge tools, like AGREE, Modified GAPI, BAGI, RAPI CaFRI and RGB12, reinforcing its white and green and sustainable nature.