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A novel HPLC method for the simultaneous determination of Prulifloxacin, Ulifloxacin, its process impurities in a tablets formulation and Enrofloxacin, used as Internal Standard, is developed. The separation was successfully carried out with a new stationary phase, HILIC, under isocratic elution mode using ammonium acetate buffer (5 mM, pH 5.8) and acetonitrile (12:88, v/v) at a flow rate of 1.0 mL/min. Column was thermostated at 25 °C (±1 °C) and 20 μL were injected for the analysis. Calibration curves were linear in the investigated range with correlation coefficient better than 0.9880, while the limit of quantifications ranged from 0.25 to 5 μg/mL, depending from the analyte. The within and between batch precision (RSD%) values ranged from 0.11% to 13.9% while within and between batch trueness (bias%) values ranged from 14.0% to -11.3%. This method for the direct determination and quantification of process impurities in pharmaceutical formulations is suitable for routine analyses in quality control laboratories and was applied to evaluate for the first time, the presence and the quantities of cited analytes in commercially available formulation.

A rapid, specific and novel gradient LC-MS method has been developed and validated for the identification and characterization of stressed degradation products (DPs) of prulifloxacin (PF) using liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS). PF was subjected to hydrolytic (acidic, alkaline and neutral), oxidation, photolytic and thermal stress, as per ICH guidelines Q1A (R2). The drug showed extensive degradation in hydrolytic and oxidative, while it was stable to thermal and photolytic stress conditions. In total, 13 DPs were formed and the chromatographic separation of drug and its DPs was achieved on a C-18 column (4.6 × 250 mm, 5 μm) using gradient elution method. All the DPs have been identified and characterized using MS(n) experiments and accurate mass measurements. The LC-MS method was validated with respect to specificity, linearity, accuracy, precision and robustness.

A rapid and simple method for determining two fluoroquinolones (FQNs), namely Ciprofloxacin and Ulifloxacin, this being the last active metabolite of Prulifloxacin, in aqueous human humor (AHH) has been developed and validated. The calibration data resulted linearly correlated in the 4-500 ng/mL concentration range with 8 ng/mL lower limit of quantification (LLOQ) for Ciprofloxacin, and 5-600 ng/mL concentration range with 6 ng/mL LOD for Ulifloxacin. The proposed analytic procedure has been validated by testing quality control sample (QCS) of AHH probed with the two FQNs at 10, 50, 500, and 1000 ng/mL concentration values. Validation of the method has been checked by accuracy and precision data of intra-day and long-term experiments. The two FQN concentrations have been measured by HPLC technique with UV detection at 278-nm wavelength for the AHH of patients to whom were supplied oral doses of FQNs (500 mg) twice in a day, within 1-24h before the surgery intervention of cataract. The average concentration of Ciprofloxacin resulted 186 ng/mL and that of Ulifloxacin 78 ng/mL. The nice quality of the proposed analytic procedure means that it may be suitable for in vivo studies of pharmacokinetics regarding these substances in the AHH medium.