Talanta 83 (2010) 110–116 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Validated liquid chromatographic-fluorescence method for the quantitation of gemifloxacin in human plasma Badraddin M.H. Al-Hadiya a,∗ , Adnan A. Khady b , Gamal A.E. Mostafa b a Clinical Pharmacy, College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451, Saudi Arabia b Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia article info Article history: Received 23 June 2010 Received in revised form 26 August 2010 Accepted 26 August 2010 Available online 22 September 2010 Keywords: Gemifloxacin HPLC Method of validation Human blood plasma Pharmacokinetic study abstract A highly selective, sensitive and rapid high performance liquid chromatographic method has been devel- oped and validated to quantify gemifloxacin in human plasma. The gemifloxacin and internal standard (ciprofloxacin) were extracted by ultrafiltration technique followed by injection into chromatographic system. Chromatographic separation was achieved on a reversed phase C 18 column with a mobile phase of acetonitrile:0.1% trifluoroacetic acid (20:80, v/v) using isocratic elution (at flow rate 1 mL min -1 ). The analytes were detected at 269 and 393 nm for excitation and emission, respectively. The assay exhibited a linear range of 25–5000 ng mL -1 for gemifloxacin in human plasma. The lower limit of detection was 10 ng mL -1 . The method was statistically validated for linearity, accuracy, precision and selectivity fol- lowing FDA guidelines. The intra- and inter-assay coefficients of variation did not exceed 7.6% deviation of the nominal concentration. The recovery of gemifloxacin from plasma was greater than 97.0%. Stabil- ity of gemifloxacin in plasma was excellent with no evidence of degradation during sample processing (auto-sampler) and at least 3 months storage in a freezer at -70 ◦ C. This validation method is applied for clinical study of the gemifloxacin in human volunteers. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Gemifloxacin [(R, S)-7-(3-aminomethyl-4-syn-methoxyimino- 1-pyrrolidinyl)-1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-1, 8- naphthyridine-3-carboxylic acid methanesulfonate] (CAS number 175463-14-6), is a recently developed fluoroquinolone antibacte- rial compound with a broad spectrum of activity (Fig. 1) [1–3]. It has shown potent antibacterial activity against clinical isolates and reference strains in both in vitro studies and experimental models of infection in animals [4,5]. It has particularly enhanced activity against gram-positive organisms, and displays fourfold higher activity than that of moxifloxacin against Streptococcus pneumoniae (minimum inhibitory concentration to inhibit 90% of isolates [MIC 90 ] is 0.03 g mL -1 ) in vitro [5]. Gemifloxacin has also shown potent activity against other major pathogens involved in respiratory tract infections, including Haemophilus influenzae and Moraxella catarrhalis and the atypical organisms, Legionella pneu- mophila, Chlamydia spp., and Mycoplasma spp. [6]. Furthermore, the compound has shown potent activity against many organisms that cause urinary tract infections. The adverse reaction profile is simi- lar to that of older members of this class [7]. The pharmacokinetic ∗ Corresponding author. Fax: +966 4677480. E-mail address: b alhadiya@yahoo.com (B.M.H. Al-Hadiya). properties of fluoroquinolone antibacterial agents have been well described [8]. Gemifloxacin is rapidly absorbed with a time to max- imum plasma concentration (T max ) of 0.5–2 h in healthy subjects and displays linear pharmacokinetics over the dosage range stud- ied (20–800 mg). The long terminal phase half-life (t 1/2 ) is 8 h after single or repeated administration. Approximately 20–30% of the administered dose is excreted unchanged in the urine and plasma protein binding of gemifloxacin is about 70% [9,10]. A few analytical methods have been published for quan- tification of gemifloxacin in human plasma using liquid chromatography–mass (LC/MS) [9–11] and –mass/mass (LC/MS/MS) [12]. However the LC/MS machine is quite expen- sive and not readily available in the most clinical, bioanalytical, educational research laboratories. A method was described by Rote and Pingle [13] for the deter- mination of gemifloxacin in spiked human plasma using liquid chromatography with UV detection. The calibration range was 30–600 ng mL -1 using liquid/liquid extraction. Liquid chromatographic methods with fluorescence detection (HPLC-FL) were developed for the determination of gemifloxacin in human serum and urine using a reversed phase, and liquid–liquid extraction (unpublished data) [9,10]. To the best of our knowledge, the following validation parame- ters: linearity, precision, accuracy, recovery and stability have not been reported combined in a single gemifloxacin HPLC-FL method 0039-9140/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2010.08.047