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A novel antibacterial quinolone was prepared with two chiral centers. For medicinal safety purposes, the four stereoisomers of the molecule are separated by HPLC using a Chiral (+)-Crownpack column (25 cm × 0.46 cm, 5.0 μm) with an eluent of 50 mM H2SO4. The values of the retention, separation, and resolution factors of RR-, SS-, RS-, and SR-stereoisomers were in the range of 1.15-7.55, 1.18 to 2.34, and 1.05 to 2.38 at 15, 20, 25, 30 and 35 °C temperatures. The values of the linearity, limits of detection, and limits of quantitation were