An Improved UPLC Method for Rapid Analysis of Levofloxacin in Human Plasma Dae-Jin Park 1,4 , Prasad B. Phapale 1 , In-Jin Jang 3 , Song Cui 1,5 , Byung-Jo Moon 5 , Jung-Eun Kim 2 , Woomi Kim 4 , Sung-Kyu Hwang 1 , Young-Ran Yoon 1,2,& 1 Clinical Trial Center, Kyungpook National University Hospital, 50 Samdok-2ga, Jung-gu, Daegu 700-721, South Korea 2 Department of Molecular Medicine, School of Medicine, Kyungpook National University, 101 Dongin-2ga, Jung-gu, Daegu 700-422, South Korea; E-Mail: yry@knu.ac.kr 3 Department of Pharmacology and Clinical Pharmacology Unit, College of Medicine and Hospital, Seoul National University, 28 Yongon-dong, Jongno-gu, Seoul, South Korea 4 Department of Pharmacology, College of Medicine, Kosin University, 34 Amnam-dong, Seo-gu, Busan, South Korea 5 Department of Biochemistry, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701, South Korea Received: 23 September 2007 / Revised: 31 March 2008 / Accepted: 21 April 2008 Online publication: 6 June 2008 Abstract A rapid, specific, and sensitive ultra-performance liquid chromatographic method for analysis of levofloxacin in human plasma has been developed and validated. Plasma sam- ples were spiked with the internal standard (enoxacin) and extracted with 10:1 (v/v) ethyl acetate–isopropanol. UPLC was performed on a 100 · 2.1 mm i.d., 1.7 lm particle, C 18 column with 88:12 (v/v) 0.4% triethylamine buffer (pH 3)–acetonitrile as mobile phase, pumped isocratically at a pressure of 11,000 psi (758 bar) and a flow-rate of 0.3 mL min 1 . Ultraviolet detection was performed at 300 nm. The retention times of levofloxacin and enoxacin were 3.4 and 2.8 min, respectively, and the run-time was 5 min. Calibration showed that response was a linear function of concentration over the range 0.05–10 lg mL 1 (r 2  0.99) and the method was validated over this range for both precision and accuracy. The relative standard deviation was <15% for both intra-day and inter-day assay (n = 5). Levofloxacin and enoxacin were stable in plasma; there was no evidence of degradation during three freeze–thaw cycles, post-preparative stability at 20 °C was 24 h, short-term stability at room temperature was 6 h, and long-term stability at 70 °C was 30 days. The method was successfully used in a study of the bioequivalence of two levo- floxacin tablet formulations in healthy volunteers. Keywords Column liquid chromatography Ultra-performance liquid chromatography Levofloxacin in human plasma Introduction Levofloxacin (CAS 100986-85-4), the L isomer of the racemic fluoroquinolone ofloxacin, has broad-spectrum antimi- crobial activity and penetrates well into cerebrospinal fluid (CSF), bone tissue, bronchial mucosa, and subcutaneous adipose tissues [1–5]. Concentrations in tissues and fluids are often in excess of plasma concentrations [5]. Accordingly, it is essential to develop a specific and sensitive assay for analysis of levoflox- acin in a variety of biological matrixes [6]. Although high-performance liquid chromatography (HPLC) is a well established analytical technique in pharmaceutical development, low effi- ciency and long separation times are its main limitations [7, 8]. Ultra-perfor- mance liquid chromatography (UPLC), which uses 1.7 lm particles at a maxi- mum operating pressure of 1,000 bar (compared with conventional HPLC on 3.5 and 5 lm particles at 400 bar), has proved to be a suitable analytical tech- nique with the advantages of increased 2008, 68, 187–192 DOI: 10.1365/s10337-008-0669-4 0009-5893/08/08 Ó 2008 Vieweg+Teubner | GWV Fachverlage GmbH Original Chromatographia 2008, 68, August (No. 3/4) 187