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A fast and easy method was validated for simultaneous determination of ciprofloxacin hydrochloride monohydrate (CP), levofloxacin hemihydrate (LV), and N-acetylcysteine (NAC) in samples. The analysis was performed on a C18 column (250 × 4.6 mm, 5 μm) (Inertsil ODS-3V) using an isocratic elution method with a mobile phase composed of 25 mM KH2 PO4 (pH 3.0) and methanol (72:28, v/v) at a flow rate of 1 mL/min. UV detection was performed at 214 nm for NAC and 293 nm for CP and LV. The method was validated for linearity, accuracy, precision (repeatability and reproducibility), specificity, sensitivity, and stability. The calibration study using several media demonstrated that the calibration curves were linear for all compounds in all media (R2 >0.9993). The limit of detection was 0.098 μg/mL for CP, 0.049 μg/mL for LV, and 0.487 μg/mL for NAC. The limit of quantification was 0.328 μg/mL for CP, 0.165 μg/mL for LV, and 1.624 μg/mL for NAC. Precision and accuracy values of the method fulfilled the required limits. All these outcomes demonstrate that the validated HPLC method is appropriate for simultaneous analysis of CP, LV, and NAC in samples for content uniformity of dry powder inhaler and permeability studies.

Determination of N-acetylcysteine in the presence of ciprofloxacin or levofloxacinin microparticulate dry powder inhalers