Estudio De Levofloxacina

Short Communications
Development and Validation of a Densitometric HPTLC Method for Quantitative Analysis of Levofloxacin in Human Plasma
Salvador Namur*, Lizbeth Cariño, and Mario González-de la Parra

Key Words
HPTLC Levofloxacin Pharmacokinetics Bioavailability

1 Introduction
Levofloxacin,(–)-(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, is an advanced-generation fluoroquinolone antibiotic. It has broad-spectrum in-vitro activity, including activity against many clinically encountered Grampositive and Gram-negative organisms [1–3]. It is an agent approved for treatment of a variety of infections at different doses and with different routes of administration [4–7]. Although several HPLC methods coupledwith different detection techniques have been reported for quantification of levofloxacin in blood plasma [8–12], a search of Medline revealed no HPTLC methods for quantification of levofloxacin in human plasma. The objective of the work discussed in this paper was, therefore to develop a simple, rapid, and selective HPTLC method for quantification of levofloxacin in human plasma.

Figure 1 Thestructures of levofloxacin (A) and amlodipine besylate (B).

plot the stock solution of levofloxacin was diluted to 300, 1000, 2000, 4000, 8000, and 10000 ng mL–1 with methanol.
2.2 Sample Preparation

2 Experimental

2.1 Chemical and Reagents

Levofloxacin (> 99%) and amlodipine besylate (> 99%), used as internal standard (IS), were obtained from Laboratorios Liomont, S.A. de C.V. (MexicoCity, Mexico). The structures of the compounds are shown in Figure 1. Methanol, dichloromethane, and chloroform (HPLC grade) and glacial acetic acid and monobasic potassium phosphate (reagent grade) were purchased from J.T. Baker (Phillipsburg, NJ, USA). Water was from a Nanopure Diamond water system (Barnstead Thermolyne, Dubuque, IA, USA). Human plasma was obtained from the Medica Sur Hospital(Mexico City). Stock solutions (0.5 mg mL–1) of levofloxacin and amlodipine besylate were prepared in methanol. To prepare the calibration
S. Namur, L. Cariño, and M. González-de la Parra, Fundación Liomont A.C. Mexico City, México, Privada Jesús del Monte 77, Cuajimalpa CP 05000, México, D.F. E-mail: snamur@liomont.com.mx

Human plasma (960 µL) was transferred to culture tubes and spiked with20 µL levofloxacin calibration solutions and with 20 µL of IS solution. KH2PO4 buffer (pH 7.4, 500 µL) was added, followed by dichloromethane (8 mL) [13, 14], and the mixture was vortex-mixed for 10 min and centrifuged at 3500 rpm for 15 min. The lower, organic, layer was isolated and evaporated to complete dryness using a TurboVapLV evaporator (Zymark, Hopkinton, MA, USA) at 40°C under a streamof nitrogen. Finally, the extract was reconstituted in 300 µL methanol and 20 µL was applied to HPTLC plates.
2.3 Chromatography

HPTLC was performed on 20 cm × 10 cm plates precoated with 0.2 mm layers of silica gel 60F254 (Macherey–Nagel, Düren, Germany). Before use the plates were washed with methanol and activated at 80°C for 60 min in a Camag (Muttenz, Switzerland) Plate Heater III. Samplesand standard were applied to the plates as 6-mm bands with a Camag ATS 4 automatic TLC sampler, using the sprayband technique (one band per concentration). The band velocity was 10 mm s–1, the first application x axis 10 mm and y axis 8 mm.
DOI: 10.1556/JPC.21.2008.3.12

Journal of Planar Chromatography 21 (2008) 3, 209–212 0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

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ShortCommunications Table 1 Method validation data for quantification of levofloxacin by HPTLC.

Method characteristic Range Coefficient of determination (R2) Correlation coefficient (R) Lower limit of quantification (LLOQ) System suitability

Value 300–10000 ng mL–1 0.997 0.998 300 ng mL–1 RSD ≤ 5%

Plates were developed to a distance of 50 mm in a 20 cm × 10 cm Camag twin-trough chamber with...