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The determination of rifampicin in pharmaceutical dosage forms using a rapid, sensitive, selective, biocompatible, and low-cost method is of vital importance in the pharmaceutical analysis field to ensure its concentration is within the effective range when administered. In this study, nitrogen-and-phosphorous-doped carbon nanodots (CNDs) were prepared using a single-step hydrothermal method with ciprofloxacin as the starting material. The CNDs showed a highly intense blue fluorescence emission centered at 450 nm, with a photoluminescence quantum yield of about 51%. Since the absorption of rifampicin was the same as the excitation spectrum of CNDs, inner filter effect (IFE) quenching occurred and it was used as a successful detection platform for the analysis of rifampicin in capsules. The detection platform showed a dynamic linear range from 1 to 100 μM (R2 = 0.9940) and the limit of detection was 0.06 μM (when S/N = 3). The average spike recovery percentage for rifampicin in the capsule samples was 100.53% (n = 5). Moreover, the sub-chronic cytotoxicity of CNDs was evaluated on healthy male mice (Balb/c) drenched with different amounts of CNDs (10 and 50 mg/kg). During this study period, no mortalities or toxicity signs were recorded in any of the experimental subjects. Based on the cytotoxicity experiment, the proposed nano-probe is considered safe and biocompatible.

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