Development and Validation of a Sensitive Method for Levofloxacin in Gingival Crevicular Fluid by HPLC Using UV-Visible Detector

Increased interest in the clinical use of antibiotics for periodontal therapy required the development of a sensitive assay for the quantitation of levofloxacin in gingival crevicular fluid (GCF). The HPLC assay employs a C18 reversed-phase Hypersil BDS column with a mobile phase composed of methanol and phosphate buffer (pH 3.5). The chromatographic separation was monitored by a UV-Visible detector with an excitation wavelength of 290 nm. The retention time of levofloxacin and ciprofloxacin were 5.55 min and 6.52 min respectively. Levofloxacin was extracted from GCF collected on capillary tubes by the addition of acetonitrile containing the internal standard ciprofloxacin, and phosphate buffer. The percentage mean extraction recovery of low, mid, and high-quality control samples was 89.53 ± 0.91 % (Mean ± SD) for levofloxacin and 91.2 ± 2.2 % for ciprofloxacin. The lowest limit of quantitation was 50 ng/ml, with a relative standard deviation of 2.56%. The interday and intraday precision at LLOQ was 3.20 ± 0.80 (mean±SD) and 3.505 ± 0.84 (mean±SD). The typical GCF volumes collected were 0.1-1 μl. The method was validated for the linear concentration range of 50-1300 ng/ml of levofloxacin on the capillary tubes. This assay for levofloxacin was shown to be an accurate, precise, and rugged method. The proposed method can be used for the estimation of levofloxacin administered as in situ gels in periodontitis.